Solution cis-Proline Conformation of IPCs Inhibitor Aureobasidin A Elucidated via NMR-Based Conformational Analysis.

Aureobasidin A (abA) is a natural depsipeptide that inhibits inositol phosphorylceramide (IPC) synthases with significant broad-spectrum antifungal activity. abA is known to have two distinct conformations in solution corresponding to trans- and cis-proline (Pro) amide bond rotamers. While the trans-Pro conformation has been studied extensively, cis-Pro conformers have remained elusive. Conformational properties of cyclic peptides are known to strongly affect both potency and cell permeability, making a comprehensive characterization of abA conformation highly desirable. Here, we report a high-resolution 3D structure of the cis-Pro conformer of aureobasidin A elucidated for the first time using a recently developed NMR-driven computational approach. This approach utilizes ForceGen's advanced conformational sampling of cyclic peptides augmented by sparse distance and torsion angle constraints derived from NMR data. The obtained 3D conformational structure of cis-Pro abA has been validated using anisotropic residual dipolar coupling measurements. Support for the biological relevance of both the cis-Pro and trans-Pro abA configurations was obtained through molecular similarity experiments, which showed a significant 3D similarity between NMR-restrained abA conformational ensembles and another IPC synthase inhibitor, pleofungin A. Such ligand-based comparisons can further our understanding of the important steric and electrostatic characteristics of abA and can be utilized in the design of future therapeutics.

Detection of lentiviral suicide gene therapy in C6 rat glioma using hyperpolarised [1-13 C]pyruvate.

Hyperpolarised [1-13 C]pyruvate MRI has shown promise in monitoring therapeutic efficacy in a number of cancers including glioma. In this study, we assessed the pyruvate response to the lentiviral suicide gene therapy of herpes simplex virus-1 thymidine kinase with the prodrug ganciclovir (HSV-TK/GCV) in C6 rat glioma and compared it with traditional MR therapy markers. Female Wistar rats were inoculated with 106 C6 glioma cells. Treated animals received intratumoural lentiviral HSV-TK gene transfers on days 7 and 8 followed by 2-week GCV therapy starting on day 10. Animals were repeatedly imaged during therapy using volumetric MRI, diffusion and relaxation mapping, as well as metabolic [1-13 C]pyruvate MRS imaging. Survival (measured as time before animals reached a humane endpoint and were euthanised) was assessed up to day 30 posttherapy. HSV-TK/GCV gene therapy lengthened the median survival time from 12 to 25 days. This was accompanied by an apparent tumour growth arrest, but no changes in diffusion or relaxation parameters in treated animals. The metabolic response was more evident in the case-by-case analysis than in the group-level analysis. Treated animals also showed a 37 ± 15% decrease (P < 0.05, n = 5) in lactate-to-pyruvate ratio between therapy weeks, whereas a 44 ± 18% increase (P < 0.05, n = 6) was observed in control animals. Hyperpolarised [1-13 C]pyruvate MRI can offer complementary metabolic information to traditional MR methods to give a more comprehensive picture of the slowly developing gene therapy response. This may benefit the detection of the successful therapy response in patients.

Cryogen-free dissolution dynamic nuclear polarization polarizer operating at 3.35 T, 6.70 T, and 10.1 T.

PURPOSE: A novel dissolution dynamic nuclear polarization (dDNP) polarizer platform is presented. The polarizer meets a number of key requirements for in vitro, preclinical, and clinical applications. METHOD: It uses no liquid cryogens, operates in continuous mode, accommodates a wide range of sample sizes up to and including those required for human studies, and is fully automated. RESULTS: It offers a wide operational window both in terms of magnetic field, up to 10.1 T, and temperature, from room temperature down to 1.3 K. The polarizer delivers a 13 C liquid state polarization for [1-13 C]pyruvate of 70%. The build-up time constant in the solid state is approximately 1200 s (20 minutes), allowing a sample throughput of at least one sample per hour including sample loading and dissolution. CONCLUSION: We confirm the previously reported strong field dependence in the range 3.35 to 6.7 T, but see no further increase in polarization when increasing the magnetic field strength to 10.1 T for [1-13 C]pyruvate and trityl. Using a custom dry magnet, cold head and recondensing, closed-cycle cooling system, combined with a modular DNP probe, and automation and fluid handling systems, we have designed a unique dDNP system with unrivalled flexibility and performance.

Simultaneous imaging of hyperpolarized [1,4-13 C2 ]fumarate, [1-13 C]pyruvate and 18 F-FDG in a rat model of necrosis in a clinical PET/MR scanner.

A co-polarization scheme for [1,4-13 C2 ]fumarate and [1-13 C]pyruvate is presented to simultaneously assess necrosis and metabolism in rats with hyperpolarized 13 C magnetic resonance (MR). The co-polarization was performed in a SPINlab polarizer. In addition, the feasibility of simultaneous positron emission tomography (PET) and MR of small animals with a clinical PET/MR scanner is demonstrated. The hyperpolarized metabolic MR and PET was demonstrated in a rat model of necrosis. The polarization and T1 of the co-polarized [1,4-13 C2 ]fumarate and [1-13 C]pyruvate substrates were measured in vitro and compared with those obtained when the substrates were polarized individually. A polarization of 36 ± 4% for fumarate and 37 ± 6% for pyruvate was obtained. We found no significant difference in the polarization and T1 values between the dual and single substrate polarization. Rats weighing about 400 g were injected intramuscularly in one of the hind legs with 200 µL of turpentine to induce necrosis. Two hours later, 13 C metabolic maps were obtained with a chemical shift imaging sequence (16 × 16) with a resolution of 3.1 × 5.0 × 25.0 mm3 . The 13 C spectroscopic images were acquired in 12 s, followed by an 8-min 18 F-2-fluoro-2-deoxy-d-glucose (18 F-FDG) PET acquisition with a resolution of 3.5 mm. [1,4-13 C2 ]Malate was observed from the tissue injected with turpentine indicating necrosis. Normal [1-13 C]pyruvate metabolism and 18 F-FDG uptake were observed from the same tissue. The proposed co-polarization scheme provides a means to utilize multiple imaging agents simultaneously, and thus to probe various metabolic pathways in a single examination. Moreover, it demonstrates the feasibility of small animal research on a clinical PET/MR scanner for combined PET and hyperpolarized metabolic MR.

Dissolution dynamic nuclear polarization of non-self-glassing agents: spectroscopy and relaxation of hyperpolarized [1-13C]acetate.

The intrinsic physicochemical properties of the sample formulation are the key factors for efficient hyperpolarization through dissolution dynamic nuclear polarization (dissolution-DNP). We provide a comprehensive characterization of the DNP process for Na-[1-(13)C]acetate selected as a model for non-self-glassing agents: the solid-state polarization dynamics of different formulations and the effect of the paramagnetic agent (trityl radical) on the pattern of polarization and the relaxation profile were extensively analyzed. We quantified the effects of the glassing agent and Gd(3+)-chelate on DNP performance. The results reported here describe the constraints of the acetate formulation useful for future studies in this field with non-self-glassing enriched molecules.

Hyperpolarized H2 O MR angiography.

PURPOSE: The aim of this study was to demonstrate that dissolution- dynamic nuclear polarization is capable of hyperpolarizing water protons and that the signal from the hyperpolarized bolus injection can be exploited in angiographic applications. METHODS: We hyperpolarized water/glycerol using dynamic nuclear polarization followed by dissolution in D2 O. RESULTS: A water (1) H signal enhancement of 77 times compared with 4.7 Tesla was obtained. This corresponds to a polarization of 3.5% for the 3.9 mol/L (1) H in D2 O . Moreover, a T1 in excess of 20 s was achieved. CONCLUSION: The use of hyperpolarized water as a contrast agent presents a new opportunity to obtain MRA images with high contrast-to-noise in a fraction of a second.

Storage of magnetization as singlet order by optimal control designed pulses.

PURPOSE: The use of hyperpolarization to enhance the sensitivity of MRI has so far been limited by the decay of the polarization through T1 relaxation. Recently, methods have been proposed that extend the lifetime of the hyperpolarization by storing the spin order in slowly relaxing singlet states. METHODS: With this aim, optimal control theory was applied to create pulses that for near-equivalent spins accomplish transfers in and out of the singlet state with maximum efficiency while ensuring robustness toward variations in the nuclear spin system Hamiltonian (chemical shift, J-couplings, B1 and B0 magnetic field inhomogeneity). RESULTS: The pulses are designed to accomplish efficient transfer with low B1 amplitude, essential for applications on preclinical and clinical MR scanners. CONCLUSION: It is demonstrated that significantly improved efficiency and robustness can be obtained within the limitations of typical MR scanner performance.

Recycling and imaging of nuclear singlet hyperpolarization.

The strong enhancement of NMR signals achieved by hyperpolarization decays, at best, with a time constant of a few minutes. Here, we show that a combination of long-lived singlet states, molecular design, magnetic field cycling, and specific radiofrequency pulse sequences allows repeated observation of the same batch of polarized nuclei over a period of 30 min and more. We report a recycling protocol in which the enhanced nuclear polarization achieved by dissolution-DNP is observed with full intensity and then returned to singlet order. MRI experiments may be run on a portion of the available spin polarization, while the remaining is preserved and made available for a later use. An analogy is drawn with a "spin bank" or "resealable container" in which highly polarized spin order may be deposited and retrieved.

Assessment of early diabetic renal changes with hyperpolarized [1-(13) C]pyruvate.

BACKGROUND: This experimental study explores a novel magnetic resonance imaging/spectroscopic (MRI/MRS) method that measures changes in renal metabolism in a diabetic rat model. This hyperpolarized metabolic MRI/MRS method allows monitoring of metabolic processes in seconds by >10 000-fold enhancement of the MR signal. The method has shown that the conversion of pyruvate to bicarbonate, i.e. pyruvate dehydrogenase (PDH) activity, is significantly altered in the myocardium already at the onset of diabetes, and the predominant Warburg effect is a valuable cancer maker via the lactate dehydrogenase (LDH) activity. We hypothesize that a similar change in PDH and LDH could be found in the early diabetic kidney. METHODS: In a streptozotocin rat model of type 1 diabetes, hyperpolarized (13) C-MRI and blood oxygenation level-dependent (1) H-MRI was employed to investigate the changes in renal metabolism in the diabetic and the control kidneys in vivo. RESULTS: The diabetic kidney showed a 149% increase in the lactate/pyruvate ratio compared with the control rat kidney, whereas the bicarbonate/pyruvate ratio was unchanged between the diabetic and the control rat kidneys, consistent with literature findings. These metabolic findings paralleled a reduced intrarenal oxygen availability as found by blood oxygenation level-dependent MRI. DISCUSSION: Hyperpolarized (13) C-MRI shows promise in the diagnosis and monitoring of early renal changes associated with diabetes, with the pyruvate/lactate ratio as an imaging biomarker for regional renal changes.

Rapid determination of biosynthetic pathways using fractional isotope enrichment and high-resolution dynamic nuclear polarization enhanced NMR.

Carbon-13 NMR has traditionally been a method of choice for the determination of metabolic pathways. Through fractional labeling, (13)C spectra allow the identification of fragments incorporated as a unit into a biosynthesized molecule. The low sensitivity of (13)C spectroscopy is an impediment to such studies, especially if compounded with an often limited availability of biosynthesized molecules. Dynamic nuclear polarization (DNP) can increase the signal-to-noise ratio in magnetic resonance by several orders of magnitude, and in combination with high-resolution spectroscopy has the potential to increase the reach of this technique for metabolic profiling. Here, we present an application of high-resolution DNP enhanced NMR to the study of the biosynthetic pathways for membrane lipids. We show that fatty acid methyl esters are readily hyperpolarized in organic solvent. The resulting spectra resolve the various structural features of the chains, including atoms near the termini, as well as unsaturated and cyclopropyl groups. Peak patterns observed in fractionally labeled samples are explained by the way feed molecules are incorporated into fatty acid chains during synthesis. Differences in multiplet intensity between samples made from glucose and acetate feedstock mixtures further reveal metabolic preferences for these precursors in the biosynthesis of the product. In addition to the present study of lipid biosynthesis, high-resolution DNP-NMR of fractionally (13)C-labeled metabolites may present itself for the rapid determination of biosynthetic pathways in various biomedical applications, especially in cases of limited availability of the products of interest.

Applications of dynamic nuclear polarization to the study of reactions and reagents in organic and biomolecular chemistry.

Nuclear Magnetic Resonance (NMR) is an important spectroscopic tool for the identification and structural characterization of molecules in chemistry and biochemistry. The most significant limitation of NMR compared to other spectroscopies is its relatively low sensitivity, which thus often requires long measurement times or large amounts of sample. A way of increasing sensitivity of single scan NMR spectra by several orders of magnitude is through hyperpolarization of nuclear spins. Dynamic nuclear polarization allows hyperpolarization of most spins in small molecules encountered in chemistry and biochemistry. NMR spectra of small amounts of samples from natural source, or from chemical synthesis can readily be acquired. Perhaps more interestingly, the availability of the entire hyperpolarized NMR signal in one single scan allows the measurement of transient processes in real time, if applied together with a stopped-flow technique. Through observation of chemical shift, different reactant and product species can be distinguished, and kinetics and mechanisms, for example in enzyme catalyzed reactions, can be elucidated. Real-time hyperpolarization-enhanced NMR is uniquely amenable to correlating atomic positions not only through space, but also over time between reactant and product species. Such correlations carry mechanistic information about a reaction, and can prove reaction pathways. Applications of this technique are emerging in different areas of chemistry concerned with rapid reactions, including not only enzymatic processes, but also chemical catalysis and protein folding.

Rapid sample injection for hyperpolarized NMR spectroscopy.

Due to its ability to enhance the signal of a single NMR scan by several orders of magnitude, solid-to-liquid state dynamic nuclear polarization (DNP) appears well suited for the analysis of minimal amounts of compounds, as well as for the study of rapid chemical reactions. A key requirement in enabling the application of DNP-NMR to typical small-molecule substances encountered in chemistry and biochemistry is the ability to obtain high-resolution spectra, while at the same time minimizing the loss of polarization due to spin relaxation between the separate steps of DNP polarization and NMR measurement. Here, we present data demonstrating the capability of measuring DNP enhanced NMR spectra of compounds with comparably short relaxation times, with only minimal line broadening attributable to the sample transfer process. We discuss the performance characteristics of a sample injection apparatus specifically designed to provide high-resolution DNP-NMR spectra of small molecule compounds.

Temporal chemical shift correlations in reactions studied by hyperpolarized nuclear magnetic resonance.

High-resolution nuclear magnetic resonance spectroscopy (NMR) has the capability of providing often unrivaled detail on molecular structure and dynamics. Through hyperpolarization, a decisive gain in signal strength can be realized, which extends the applicability of NMR to the investigation of rapid processes far from equilibrium. The progress of irreversible chemical and biochemical reactions can be followed by hyperpolarized NMR with relative ease, within an observable window encompassing the subsecond to second time scales. Here, we present a scheme that uses real-time, hyperpolarization enhanced NMR to make temporal correlations accessible in addition to simply monitoring reaction progress. Since nuclear spin states can be preserved even if the spin carrying atoms directly participate in a reaction, it becomes possible to correlate the positions of these atoms between the reactant and the product species, over time. We demonstrate the application of this technique to the Grignard addition of methylmagnesium bromide to 3-methylbenzophenone. The same experiment may be used for the determination of mechanisms and intermediate states in non-equilibrium processes in fields as varied as organic chemistry, enzymology, or protein folding.

Chemical shift correlations from hyperpolarized NMR by off-resonance decoupling.

Nuclear magnetic resonance, through observation of chemical shift, allows the separate identification of each atom in a molecule. Thus, NMR spectra impart an often unrivaled wealth of information on molecular structure. A particular advantage of NMR spectroscopy is the ability to record multidimensional spectra, which provide correlations between atoms. When compared to other techniques, such as optical spectroscopy, the acquisition of NMR spectra is however an insensitive process, requiring samples of high concentration and long acquisition times. Recently, it has been demonstrated that dynamic nuclear polarization, a hyperpolarization technique, can increase the NMR signal by several orders of magnitude. Here, we present a robust method that allows recording two-dimensional chemical shift correlations from such hyperpolarized molecules. The method makes use of an apparent scaling of the scalar coupling observed on one type of atom, when an off-resonance decoupling field is applied to another type of atom. Thus, two-dimensional chemical shift correlations can be read directly from a small number of scans acquired using a hyperpolarized sample. Due to the ease of implementing this technique on commercial hyperpolarization and NMR equipment, it appears ideally suited for routine application, for example, to obtain carbon-proton chemical shift correlations in organic molecules.

Large dose hyperpolarized water with dissolution-DNP at high magnetic field.

We demonstrate a method for the preparation of hyperpolarized water by dissolution Dynamic Nuclear Polarization at high magnetic field. Protons were polarized at 6.7T and 1.1K to >70% with frequency modulated microwave irradiation at 188GHz. 97.2±0.7% of the radical was extracted from the sample in the dissolution in a two-phase system. 16±1mL of 5.0M 1H in D2O with a polarization of 13.0±0.9% in the liquid state was obtained, corresponding to an enhancement factor of 4000±300 compared to the thermal equilibrium at 9.4T and 293K. A longitudinal relaxation time constant of 16±1s was measured. The sample was polarized and dissolved in a fluid path compatible with clinical polarizers. The volume of hyperpolarized water produced by this method enables angiography and perfusion measurements in large animals, as well as NMR experiments for studies of e.g. proton exchange and polarization transfer to other nuclei.

Waveguide transition with vacuum window for multiband dynamic nuclear polarization systems.

A low loss waveguide transition section and oversized microwave vacuum window covering several frequency bands (94 GHz, 140 GHz, 188 GHz) is presented. The transition is compact and was optimized for multiband Dynamic Nuclear Polarization (DNP) systems in a full-wave simulator. The window is more broadband than commercially available windows, which are usually optimized for single band operation. It is demonstrated that high-density polyethylene with urethane adhesive can be used as a low loss microwave vacuum window in multiband DNP systems. The overall assembly performance and dimensions are found using full-wave simulations. The practical aspects of the window implementation in the waveguide are discussed. To verify the design and simulation results, the window is tested experimentally at the three frequencies of interest.

Enhanced performance large volume dissolution-DNP.

A systematic study of the performance of the dissolution process in dissolution-DNP is presented. A relatively simple set of modifications is made to the standard Hypersense dissolution system to enable polarization of large volume samples. These consist of a large volume sample cup along with supporting modifications to the dissolution head and related components. Additional modifications were made to support the mapping of the temperature/pressure space of the dissolution process as well as enabling the use of large volumes of solvent and improving the robustness of the system. No loss of polarization was observed as sample size was increased to the 1 g capacity of the large volume cup and for a dilution factor as low as 1:10.

Formulation and utilization of choline based samples for dissolution dynamic nuclear polarization.

Hyperpolarization by the dissolution dynamic nuclear polarization (DNP) technique permits the generation of high spin polarization of solution state. However, sample formulation for dissolution-DNP is often difficult, as concentration and viscosity must be optimized to yield a dissolved sample with sufficient concentration, while maintaining polarization during the dissolution process. The unique chemical properties of choline permit the generation of highly soluble salts as well as deep eutectic mixtures with carboxylic acids and urea. We describe the formulation of these samples and compare their performance to more traditional sample formulations. Choline yields stable samples with exceptional polarization performance while simultaneously offering the capability to easily remove the choline after dissolution, perform experiments with the hyperpolarized choline, or anything in between.

Spontaneous emission of NMR signals in hyperpolarized proton spin systems.

Hyperpolarization of nuclear spins is gaining increasing interest as a tool for improving the signal-to-noise ratio of NMR and MRI. While in principle, hyperpolarized samples are amenable to the same or similar experiments as are used in conventional NMR, the large spin polarization may give rise to unexpected effects. Here, spontaneous emission of signal was observed from proton spin systems, which were hyperpolarized to negative spin temperature by dynamic nuclear polarization (DNP). An unexpected feature of these emissions is that, without any radio-frequency excitation, multiple beats arise that cannot be explained by the Bloch equations with radiation damping. However, we show that a simple modification to these equations, which takes into account an additional supply of hyperpolarized magnetization from a reservoir outside of the active detection region, can phenomenologically describe the observed signal. The observed effect demonstrates that even well-known mechanisms of spin evolution can give rise to unexpected effects when working with hyperpolarized samples, which may need to be addressed through the development of new experimental techniques.