Reversible Parahydrogen Induced Hyperpolarization of 15 N in Unmodified Amino Acids Unraveled at High Magnetic Field.

Amino acids (AAs) and ammonia are metabolic markers essential for nitrogen metabolism and cell regulation in both plants and humans. NMR provides interesting opportunities to investigate these metabolic pathways, yet lacks sensitivity, especially in case of 15 N. In this study, spin order embedded in p-H2 is used to produce on-demand reversible hyperpolarization in 15 N of pristine alanine and ammonia under ambient protic conditions directly in the NMR spectrometer. This is made possible by designing a mixed-ligand Ir-catalyst, selectively ligating the amino group of AA by exploiting ammonia as a strongly competitive co-ligand and preventing deactivation of Ir by bidentate ligation of AA. The stereoisomerism of the catalyst complexes is determined by hydride fingerprinting using 1 H/D scrambling of the associated N-functional groups on the catalyst (i.e., isotopological fingerprinting), and unravelled by 2D-ZQ-NMR. Monitoring the transfer of spin order from p-H2 to 15 N nuclei of ligated and free alanine and ammonia targets using SABRE-INEPT with variable exchange delays pinpoints the monodentate elucidated catalyst complexes to be most SABRE active. Also RF-spin locking (SABRE-SLIC) enables transfer of hyperpolarization to 15 N. The presented high-field approach can be a valuable alternative to SABRE-SHEATH techniques since the obtained catalytic insights (stereochemistry and kinetics) will remain valid at ultra-low magnetic fields.

Amplified Overhauser DNP with Selective Deuteration: Attenuation of Double-Quantum Cross-Relaxation.

We recently used selective 2H labeling of BDPA to investigate the Overhauser Effect (OE) dynamic nuclear polarization (DNP) mechanism in insulating solids doped with 1,3-bis(diphenylene)-2-phenylallyl (BDPA), and established that the α and γ 1H spins on the fluorene rings are responsible for generating a zero quantum (ZQ) mediated positive bulk polarization. Here, we establish that the phenyl 1H spins relax via double-quantum (DQ) processes and therefore contribute negative enhancements which attenuate the OE-DNP. With measurements at different magnetic field strengths, we show that phenyl-d5-BDPA offers >50% improvement in OE-DNP enhancement compared to h21-BDPA attaining a maximum of ∼90 at 14.1 T and 5 kHz MAS, the highest observed OE-DNP enhancement to date under these conditions. The approach may be utilized to optimize other polarizing agents exhibiting an OE, an important DNP mechanism with a favorable field and spinning frequency dependence.

Isotopological Fingerprinting Using 1H/D Scrambling Identifies the Stereochemistry of Hyperpolarization Catalysts Transferring Spin Polarization from Parahydrogen to Substrates Using Signal Amplification by Reversible Exchange.

Hyperpolarization using signal amplification by reversible exchange (SABRE) relies on target molecules and parahydrogen coordinating to a transition metal catalyst. Identification of this coordinated state becomes increasingly important, especially since bio-relevant targets such as pyruvate and amino acids exhibiting multiple binding sites are becoming compatible with SABRE. In this report, we present a fingerprinting method to discriminate and identify ligand binding sites without requiring the presence of a sensitive or isotope-labeled heteroatom. Adding a small concentration of protons to a deuterated medium, spontaneous 1H/D scrambling of exchangeable protons encodes the ligands each with an isotopological fingerprint. By use of rapid 2D zero quantum NMR, the binding sites are decoded from the hydrides in less than a minute. The new methodology is explained and demonstrated on Ir mixed complexes with pyridine, benzylamine, and ammonia as common N-functional ligands.

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

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

Long-Term Generation of Longitudinal Spin Order Controlled by Ammonia Ligation Enables Rapid SABRE Hyperpolarized 2D NMR.

The front cover artwork front cover artwork is provided by NMRCoRe, the Flemish NMR/X-Ray platform for Convergence Research and was designed by Ir. Ewoud Vaneeckhaute and Dr. Eric Breynaert. The image shows the reciprocity between parahydrogen, deuterated ammonia and iridium allowing for hyperpolarized 2D NMR via long-term availability of longitudinal spin order. Read the full text of the Article at 10.1002/cphc.202100079.

Long-Term Generation of Longitudinal Spin Order Controlled by Ammonia Ligation Enables Rapid SABRE Hyperpolarized 2D NMR.

Symmetry breaking of parahydrogen using iridium catalysts converts singlet spin order into observable hyperpolarization. In this contribution, iridium catalysts are designed to exhibit asymmetry in their hydrides, regulated by in situ generation of deuterated ammonia governed by ammonium buffers. The concentrations of ammonia (N) and pyridine (P) provide a handle to generate a variety of stereo-chemically asymmetric N-heterocyclic carbene iridium complexes, ligating either [3xP], [2xP;N], [P;2xN] or [3xN] in an octahedral SABRE type configuration. The non-equivalent hydride positions, in correspondence with the ammonium buffer solutions, enables to extend singlet-triplet or S⟩→T0⟩ mixing at high magnetic field and experimentally induce prolonged generation of non-equilibrium longitudinal two-spin order. This long-lasting magnetization can be exploited in hyperpolarized 2D-OPSY-COSY experiments providing direct structural information on the catalyst using a single contact with parahydrogen. Separately, field cycling revealed hyperpolarization properties in low-field conditions. Controlling catalyst stereochemistry by introducing small and deuterated ligands, such as deuterated ammonia, simplifies the spin-system. This is shown to unify experimental and theoretically derived field-sweep experiments for four-spin systems.

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

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

Hyperpolarized NMR Metabolomics at Natural 13C Abundance.

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

DOSY Analysis of Micromolar Analytes: Resolving Dilute Mixtures by SABRE Hyperpolarization.

DOSY is an NMR spectroscopy technique that resolves resonances according to the analytes' diffusion coefficients. It has found use in correlating NMR signals and estimating the number of components in mixtures. Applications of DOSY in dilute mixtures are, however, held back by excessively long measurement times. We demonstrate herein, how the enhanced NMR sensitivity provided by SABRE hyperpolarization allows DOSY analysis of low-micromolar mixtures, thus reducing the concentration requirements by at least 100-fold.

Communication: Dissolution DNP reveals a long-lived deuterium spin state imbalance in methyl groups.

We report the generation and observation of long-lived spin states in deuterated methyl groups by dissolution DNP. These states are based on population imbalances between manifolds of spin states corresponding to irreducible representations of the C3v point group and feature strongly dampened quadrupolar relaxation. Their lifetime depends on the activation energies of methyl group rotation. With dissolution DNP, we can reduce the deuterium relaxation rate by a factor up to 20, thereby extending the experimentally available time window. The intrinsic limitation of NMR spectroscopy of quadrupolar spins by short relaxation times can thus be alleviated.

Correction: Low-field thermal mixing in [1-(13)C] pyruvic acid for brute-force hyperpolarization.

Correction for 'Low-field thermal mixing in [1-(13)C] pyruvic acid for brute-force hyperpolarization' by David T. Peat et al., Phys. Chem. Chem. Phys., 2016, 18, 19173-19182.

Low-field thermal mixing in [1-(13)C] pyruvic acid for brute-force hyperpolarization.

We detail the process of low-field thermal mixing (LFTM) between (1)H and (13)C nuclei in neat [1-(13)C] pyruvic acid at cryogenic temperatures (4-15 K). Using fast-field-cycling NMR, (1)H nuclei in the molecule were polarized at modest high field (2 T) and then equilibrated with (13)C nuclei by fast cycling (∼300-400 ms) to a low field (0-300 G) that activates thermal mixing. The (13)C NMR spectrum was recorded after fast cycling back to 2 T. The (13)C signal derives from (1)H polarization via LFTM, in which the polarized ('cold') proton bath contacts the unpolarised ('hot') (13)C bath at a field so low that Zeeman and dipolar interactions are similar-sized and fluctuations in the latter drive (1)H-(13)C equilibration. By varying mixing time (tmix) and field (Bmix), we determined field-dependent rates of polarization transfer (1/τ) and decay (1/T1m) during mixing. This defines conditions for effective mixing, as utilized in 'brute-force' hyperpolarization of low-γ nuclei like (13)C using Boltzmann polarization from nearby protons. For neat pyruvic acid, near-optimum mixing occurs for tmix∼ 100-300 ms and Bmix∼ 30-60 G. Three forms of frozen neat pyruvic acid were tested: two glassy samples, (one well-deoxygenated, the other O2-exposed) and one sample pre-treated by annealing (also well-deoxygenated). Both annealing and the presence of O2 are known to dramatically alter high-field longitudinal relaxation (T1) of (1)H and (13)C (up to 10(2)-10(3)-fold effects). Here, we found smaller, but still critical factors of ∼(2-5)× on both τ and T1m. Annealed, well-deoxygenated samples exhibit the longest time constants, e.g., τ∼ 30-70 ms and T1m∼ 1-20 s, each growing vs. Bmix. Mixing 'turns off' for Bmix > ∼100 G. That T1m≫τ is consistent with earlier success with polarization transfer from (1)H to (13)C by LFTM.

Transport and imaging of brute-force (13)C hyperpolarization.

We demonstrate transport of hyperpolarized frozen 1-(13)C pyruvic acid from its site of production to a nearby facility, where a time series of (13)C images was acquired from the aqueous dissolution product. Transportability is tied to the hyperpolarization (HP) method we employ, which omits radical electron species used in other approaches that would otherwise relax away the HP before reaching the imaging center. In particular, we attained (13)C HP by 'brute-force', i.e., using only low temperature and high-field (e.g., T<∼2K and B∼14T) to pre-polarize protons to a large Boltzmann value (∼0.4% (1)H polarization). After polarizing the neat, frozen sample, ejection quickly (<1s) passed it through a low field (B<100G) to establish the (1)H pre-polarization spin temperature on (13)C via the process known as low-field thermal mixing (yielding ∼0.1% (13)C polarization). By avoiding polarization agents (a.k.a. relaxation agents) that are needed to hyperpolarize by the competing method of dissolution dynamic nuclear polarization (d-DNP), the (13)C relaxation time was sufficient to transport the sample for ∼10min before finally dissolving in warm water and obtaining a (13)C image of the hyperpolarized, dilute, aqueous product (∼0.01% (13)C polarization, a >100-fold gain over thermal signals in the 1T scanner). An annealing step, prior to polarizing the sample, was also key for increasing T1∼30-fold during transport. In that time, HP was maintained using only modest cryogenics and field (T∼60K and B=1.3T), for T1((13)C) near 5min. Much greater time and distance (with much smaller losses) may be covered using more-complete annealing and only slight improvements on transport conditions (e.g., yielding T1∼5h at 30K, 2T), whereas even intercity transfer is possible (T1>20h) at reasonable conditions of 6K and 2T. Finally, it is possible to increase the overall enhancement near d-DNP levels (i.e., 10(2)-fold more) by polarizing below 100mK, where nanoparticle agents are known to hasten T1 buildup by 100-fold, and to yield very little impact on T1 losses at temperatures relevant to transport.

Brute-Force Hyperpolarization for NMR and MRI.

Hyperpolarization (HP) of nuclear spins is critical for ultrasensitive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). We demonstrate an approach for >1500-fold enhancement of key small-molecule metabolites: 1-(13)C-pyruvic acid, 1-(13)C-sodium lactate, and 1-(13)C-acetic acid. The (13)C solution NMR signal of pyruvic acid was enhanced 1600-fold at B = 1 T and 40 °C by pre-polarizing at 14 T and ∼2.3 K. This "brute-force" approach uses only field and temperature to generate HP. The noted 1 T observation field is appropriate for benchtop NMR and near the typical 1.5 T of MRI, whereas high-field observation scales enhancement as 1/B. Our brute-force process ejects the frozen, solid sample from the low-T, high-B polarizer, passing it through low field (B < 100 G) to facilitate "thermal mixing". That equilibrates (1)H and (13)C in hundreds of milliseconds, providing (13)C HP from (1)H Boltzmann polarization attained at high B/T. The ejected sample arrives at a room-temperature, permanent magnet array, where rapid dissolution with 40 °C water yields HP solute. Transfer to a 1 T NMR system yields (13)C signals with enhancements at 80% of ideal for noted polarizing conditions. High-resolution NMR of the same product at 9.4 T had consistent enhancement plus resolution of (13)C shifts and J-couplings for pyruvic acid and its hydrate. Comparable HP was achieved with frozen aqueous lactate, plus notable enhancement of acetic acid, demonstrating broader applicability for small-molecule NMR and metabolic MRI. Brute-force avoids co-solvated free-radicals and microwaves that are essential to competing methods. Here, unadulterated samples obviate concerns about downstream purity and also exhibit slow solid-state spin relaxation, favorable for transporting HP samples.

NMR studies of the chiral selectivity of self-assembled guanosinemonophosphate.

Among nucleobases in RNA and DNA, only guanine exhibits reversible self-assembly through Hoogsteen hydrogen bonding. Resulting "G-tetrads" are building blocks of higher-order structures of guanosine compounds, including GMP. These can further associate to form chiral, columnar assemblies, stabilized by a centrally located cation such as K(+). Here, we detail chiral selectivity of GMP under conditions for assembly, as previously used to separate enantiomeric solutes by capillary electrophoresis (CE). Using (1)H NMR, we track multisite chemical-shift responses vs the concentration of various chiral solutes with GMP. Shift changes in both GMP and the solutes are consistent with a structural model in which grooves of transiently assembled GMP are templates for chiral selection. This can also explain a previously noted superiority of selection for solutes with extended ring systems projecting from the chiral center. Here, site-by-site NMR shift changes (and their enantiomeric differential) are strongest at the ring extremities in d- vs l-tryptophan and R- vs S-BNPA. This is consistent with the fact that, for one enantiomer of each pair, steric hindrance prevents closest approach into assembled GMP. For smaller solutes (d/l-Trp and d/l-Phe), NMR reveals only slight (Tyr) or no (Phe) chiral discrimination, consistent with their failure to resolve in CE through GMP media. We present molecular models to visualize and evaluate a proposed mode of selection.

Fat-referenced MR thermometry in the breast and prostate using IDEAL.

PURPOSE: To demonstrate a three-echo fat-referenced MR thermometry technique that estimates and corrects for time-varying phase disturbances in heterogeneous tissues. MATERIALS AND METHODS: Fat protons do not exhibit a temperature-dependent frequency shift. Fat-referenced thermometry methods exploit this insensitivity and use the signal from fat to measure and correct for magnetic field disturbances. In this study, we present a fat-referenced method that uses interpolation of the fat signal to correct for phase disturbances in fat free regions. Phantom and ex vivo tissue cool-down experiments were performed to evaluate the accuracy of this method in the absence of motion. Non-heated in vivo imaging of the breast and prostate was performed to demonstrate measurement robustness in the presence of systemic and motion-induced field disturbances. Measurement accuracy of the method was compared to conventional proton resonance frequency shift MR thermometry. RESULTS: In the ex vivo porcine tissue experiment, maximum measurement error of the fat-referenced method was reduced 42% from 3.3 to 1.9°C when compared to conventional MR thermometry. In the breasts, measurement errors were reduced by up to 70% from 6.4 to 1.9°C. CONCLUSION: Ex vivo and in vivo results show that the proposed method reduces measurement errors in the heterogeneous tissue experiments when compared to conventional MR thermometry.

Coherent NMR Stark spectroscopy.

We demonstrate phase-coherent Stark effects from a radiofrequency E field at twice the NMR frequency (2ω(0)) of (69)Ga in GaAs. The 2ω(0) phase (ϕ(E)) selects component responses from the nuclear quadrupole Hamiltonian (H(Q)). This is possible by synchronizing few-µs 2ω(0) pulses with an NMR line-narrowing sequence, which averages the Stark interaction to dominate spectra on a background with 10(3)× enhanced resolution. Spectra vs ϕ(E) reveal relative sizes of tensorial factors in H(Q). Comparative modeling and numerical simulations evaluate spectral features unexplained by average Hamiltonian theory, and suggest improvements for quantitative calibration of individual response components. Application of this approach to bulk samples is of value to define Stark responses that may later be used to interrogate the internal electrostatics of structured samples.

Quantitative calibration of radiofrequency NMR Stark effects.

Nuclear magnetic resonance (NMR) Stark responses can occur in quadrupolar nuclei for an electric field oscillating at twice the usual NMR frequency (2ω(0)). Calibration of responses to an applied E field is needed to establish nuclear spins as probes of native E fields within material and molecular systems. We present an improved approach and apparatus for accurate measurement of quadrupolar Stark effects. Updated values of C(14) (the response parameter in cubic crystals) were obtained for both (69)Ga and (75)As in GaAs. Keys to improvement include a modified implementation of voltage dividers to assess the 2ω(0) amplitude, |E|, and the stabilization of divider response by reduction of stray couplings in 2ω(0) circuitry. Finally, accuracy was enhanced by filtering sets of |E| through a linear response function that we established for the radiofrequency amplifier. Our approach is verified by two types of spectral results. Steady-state 2ω(0) excitation to presaturate NMR spectra yielded C(14) = (2.59 ± 0.06) × 10(12) m(-1) for (69)Ga at room-temperature and 14.1 T. For (75)As, we obtained (3.1 ± 0.1) × 10(12) m(-1). Both values reconcile with earlier results from 77 K and below 1 T, whereas current experiments are at room temperature and 14.1 T. Finally, we present results where few-microsecond pulses of the 2ω(0) field induced small (tens of Hz) changes in high-resolution NMR line shapes. There too, spectra collected vs |E| agree with the model for response, further establishing the validity of our protocols to specify |E|.

Enhancing time-suspension sequences for the measurement of weak perturbations.

We detail key features for implementation of time-suspension multiple-pulse line-narrowing sequences. This sequence class is designed to null the average Hamiltonian (H¯(°)) over the period of the multiple-pulse cycle, typically to provide for high-resolution isolation of evolution from a switched interaction, such as field gradients for imaging or small sample perturbations. Sequence designs to further ensure null contributions from correction terms (H¯((¹)) and H¯(²)) of the Magnus expansion are also well known, as are a variety of approaches to second averaging, the process by which diagonal content is incorporated in H¯(°) to truncate unwanted terms. In spite of such designs, we observed spin evolution not explicable by H¯(°) using 16-, 24- and 48-pulse time-suspension sequences. We found three approaches to effectively remove artifacts that included splitting of the lineshape into unexpected multiplets as well as chirped evolution. The noted approaches are simultaneously compatible for combination of their benefits. The first ensures constant power deposition from RF excitation as the evolution period is incremented. This removes chirping and allows more effective 2nd averaging. Two schemes for the latter are evaluated: the noted introduction of a diagonal term in H¯(°), and phase-stepping the line-narrowing sequence on successive instances during the evolution period. Either of these was sufficient to remove artifactual splittings and to further enhance resolution, while in combination enhancements were maintained. Finally, numerical simulations provide evidence that our experimental line-narrowing results with 75As in crystalline GaAs approach performance limits of idealized sequences (e.g., with ideal square pulses, etc.). The three noted experimental techniques should likewise benefit ultimate implementation with switched interactions and corresponding new error contributions, which place further demand on sequence performance.

Radiofrequency quadrupolar NMR stark spectroscopy: steady state response calibration and tensorial mapping.

Radiofrequency electric (E) fields oscillating at twice the usual NMR frequency (2ω(0)) can induce double-quantum transitions in quadrupolar nuclei, an NMR Stark effect. Characterization of such is of interest to aid understanding of electrostatic effects in NMR spectra. Calibration of Stark responses to an applied electric field may also be used to assess native fields within molecules and materials. We present high-field (14.1 T), room-temperature NMR experiments to calibrate the 2ω(0) Stark response in crystalline GaAs. This system presents an important test of current techniques and conditions, as historical studies at low field (500-900 mT) and low temperature (77 K) provide a basis for comparison. Our measurements of steady state response reveal the quadrupolar Stark tuning rate for (69)Ga in this material. The value, ß(Q) = (11.5 ± 0.1) × 10(12) m(-1), is 3.6 times larger than the most-reliable prior result. In the process, we also uncovered a previously unobserved double-quantum steady state coherence. It appears as a completely separable dispersive signal component in quadrature-detected presaturation spectra versus offset from 2ω(0). The new component may eventually afford an independent route to calibrating ß(Q). Finally, we demonstrated exceptional agreement with theory of the orientation-dependent Stark response for rotation of the sample relative to B(0) over a range of 90° and for E-field amplitudes from 30-180 V/cm.