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Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at 13C polarization levels of 30-45%.
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Técnicas Biosensibles , Espectroscopía de Resonancia Magnética con Carbono-13 , Fumaratos/aislamiento & purificación , Fumaratos/metabolismo , Imagen Molecular/métodos , Agua/química , SolucionesRESUMEN
We present a versatile method for the preparation of hyperpolarized [1-13C]fumarate as a contrast agent for preclinical in vivo MRI, using parahydrogen-induced polarization (PHIP). To benchmark this process, we compared a prototype PHIP polarizer to a state-of-the-art dissolution dynamic nuclear polarization (d-DNP) system. We found comparable polarization, volume, and concentration levels of the prepared solutions, while the preparation effort is significantly lower for the PHIP process, which can provide a preclinical dose every 10 min, opposed to around 90 min for d-DNP systems. With our approach, a 100 mM [1-13C]-fumarate solution of volumes up to 3 mL with 13-20% 13C-hyperpolarization after purification can be produced. The purified solution has a physiological pH, while the catalyst, the reaction side products, and the precursor material concentrations are reduced to nontoxic levels, as confirmed in a panel of cytotoxicity studies. The in vivo usage of the hyperpolarized fumarate as a perfusion agent in healthy mice and the metabolic conversion of fumarate to malate in tumor-bearing mice developing regions with necrotic cell death is demonstrated. Furthermore, we present a one-step synthesis to produce the 13C-labeled precursor for the hydrogenation reaction with high yield, starting from 13CO2 as a cost-effective source for 13C-labeled compounds.
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Fumaratos , Imagen por Resonancia Magnética , Ratones , Animales , Espectroscopía de Resonancia Magnética , Imagen por Resonancia Magnética/métodos , Hidrogenación , Medios de ContrasteRESUMEN
Zero- to ultralow-field nuclear magnetic resonance is a modality of magnetic resonance experiment which does not require strong superconducting magnets. Contrary to conventional high-field nuclear magnetic resonance, it has the advantage of allowing high-resolution detection of nuclear magnetism through metal as well as within heterogeneous media. To achieve high sensitivity, it is common to couple zero-field nuclear magnetic resonance with hyperpolarization techniques. To date, the most common technique is parahydrogen-induced polarization, which is only compatible with a small number of compounds. In this article, we establish dissolution dynamic nuclear polarization as a versatile method to enhance signals in zero-field nuclear magnetic resonance experiments on sample mixtures of [13C]sodium formate, [1-13C]glycine, and [2-13C]sodium acetate, and our technique is immediately extendable to a broad range of molecules with >1 s relaxation times. We find signal enhancements of up to 11,000 compared with thermal prepolarization in a 2 T permanent magnet. To increase the signal in future experiments, we investigate the relaxation effects of the TEMPOL radicals used for the hyperpolarization process at zero- and ultralow-fields.
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Imagen por Resonancia Magnética , Solubilidad , Espectroscopía de Resonancia Magnética/métodos , Imagen por Resonancia Magnética/métodosRESUMEN
We demonstrate that enzyme-catalyzed reactions can be observed in zero- and low-field NMR experiments by combining recent advances in parahydrogen-based hyperpolarization methods with state-of-the-art magnetometry. Specifically, we investigated two model biological processes: the conversion of fumarate into malate, which is used in vivo as a marker of cell necrosis, and the conversion of pyruvate into lactate, which is the most widely studied metabolic process in hyperpolarization-enhanced imaging. In addition to this, we constructed a microfluidic zero-field NMR setup to perform experiments on microliter-scale samples of [1-13C]fumarate in a lab-on-a-chip device. Zero- to ultralow-field (ZULF) NMR has two key advantages over high-field NMR: the signals can pass through conductive materials (e.g., metals), and line broadening from sample heterogeneity is negligible. To date, the use of ZULF NMR for process monitoring has been limited to studying hydrogenation reactions. In this work, we demonstrate this emerging analytical technique for more general reaction monitoring and compare zero- vs low-field detection.
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Imagen por Resonancia Magnética , Ácido Pirúvico , Espectroscopía de Resonancia Magnética/métodos , Imagen por Resonancia Magnética/métodos , Hidrogenación , Ácido Pirúvico/metabolismo , FumaratosRESUMEN
There is a fundamental issue with the use of dynamic nuclear polarization (DNP) to enhance nuclear spin polarization: the same polarizing agent (PA) needed for DNP is also responsible for shortening the lifetime of the hyperpolarization. As a result, long-term storage and transport of hyperpolarized samples is severely restricted and the apparatus for DNP is necessarily located near or integrated with the apparatus using the hyperpolarized spins. In this paper, we demonstrate that naphthalene single crystals can serve as a long-lived reservoir of proton polarization that can be exploited to enhance signals in benchtop and high-field NMR of target molecules in solution at a site 300 km away by a factor of several thousand. The naphthalene protons are polarized using short-lived optically excited triplet states of pentacene instead of stable radicals. In the absence of optical excitation, the electron spins remain in a singlet ground state, eliminating the major pathway of nuclear spin-lattice relaxation. The polarization decays with a time constant of about 50 h at 80 K and 0.5 T or above 800 h at 5 K and 20 mT. A module based on a Halbach array yielding a field of 0.75 T and a conventional cryogenic dry shipper, operating at liquid nitrogen temperature, allows storage and long distance transport of the polarization to a remote laboratory, where the polarization of the crystal is transferred after dissolution to a target molecule of choice by intermolecular cross-relaxation. The procedure has been executed repeatedly and has proven to be reliable and robust.
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The ever-increasing demand for high-capacity rechargeable batteries highlights the need for sensitive and accurate diagnostic technology for determining the state of a cell, for identifying and localizing defects, and for sensing capacity loss mechanisms. Here, we leverage atomic magnetometry to map the weak induced magnetic fields around Li-ion battery cells in a magnetically shielded environment. The ability to rapidly measure cells nondestructively allows testing even commercial cells in their actual operating conditions, as a function of state of charge. These measurements provide maps of the magnetic susceptibility of the cell, which follow trends characteristic for the battery materials under study upon discharge. In particular, hot spots of charge storage are identified. In addition, the measurements reveal the capability to measure transient internal current effects, at a level of µA, which are shown to be dependent upon the state of charge. These effects highlight noncontact battery characterization opportunities. The diagnostic power of this technique could be used for the assessment of cells in research, quality control, or during operation, and could help uncover details of charge storage and failure processes in cells.
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The HYPNOESYS method (Hyperpolarized NOE System), which relies on the dissolution of optically polarized crystals, has recently emerged as a promising approach to enhance the sensitivity of NMR spectroscopy in the solution state. However, HYPNOESYS is a single-shot method that is not generally compatible with multidimensional NMR. Here we show that 2D NMR spectra can be obtained from HYPNOESYS-polarized samples, using single-scan acquisition methods. The approach is illustrated with a mixture of terpene molecules and a benchtop NMR spectrometer, paving the way to a sensitive, information-rich and affordable analytical method.
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Nuclear spin hyperpolarization provides a promising route to overcome the challenges imposed by the limited sensitivity of nuclear magnetic resonance. Here we demonstrate that dissolution of spin-polarized pentacene-doped naphthalene crystals enables transfer of polarization to target molecules via intermolecular cross-relaxation at room temperature and moderate magnetic fields (1.45 T). This makes it possible to exploit the high spin polarization of optically polarized crystals, while mitigating the challenges of its transfer to external nuclei. With this method, we inject the highly polarized mixture into a benchtop NMR spectrometer and observe the polarization dynamics for target 1H nuclei. Although the spectra are radiation damped due to the high naphthalene magnetization, we describe a procedure to process the data to obtain more conventional NMR spectra and extract the target nuclei polarization. With the entire process occurring on a time scale of 1 min, we observe NMR signals enhanced by factors between -200 and -1730 at 1.45 T for a range of small molecules.
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We report the results of an experimental search for ultralight axionlike dark matter in the mass range 162-166 neV. The detection scheme of our Cosmic Axion Spin Precession Experiment is based on a precision measurement of ^{207}Pb solid-state nuclear magnetic resonance in a polarized ferroelectric crystal. Axionlike dark matter can exert an oscillating torque on ^{207}Pb nuclear spins via the electric dipole moment coupling g_{d} or via the gradient coupling g_{aNN}. We calibrate the detector and characterize the excitation spectrum and relaxation parameters of the nuclear spin ensemble with pulsed magnetic resonance measurements in a 4.4 T magnetic field. We sweep the magnetic field near this value and search for axionlike dark matter with Compton frequency within a 1 MHz band centered at 39.65 MHz. Our measurements place the upper bounds |g_{d}|<9.5×10^{-4} GeV^{-2} and |g_{aNN}|<2.8×10^{-1} GeV^{-1} (95% confidence level) in this frequency range. The constraint on g_{d} corresponds to an upper bound of 1.0×10^{-21} e cm on the amplitude of oscillations of the neutron electric dipole moment and 4.3×10^{-6} on the amplitude of oscillations of CP-violating θ parameter of quantum chromodynamics. Our results demonstrate the feasibility of using solid-state nuclear magnetic resonance to search for axionlike dark matter in the neV mass range.
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The field of zero- to ultralow-field (ZULF) nuclear magnetic resonance (NMR) is currently experiencing rapid growth, owing to progress in optical magnetometry and attractive features of ZULF-NMR such as low hardware cost and excellent spectral resolution achieved under ZULF conditions. In this work, an approach is proposed and demonstrated for simultaneous acquisition of ZULF-NMR spectra of individual 13C-containing isotopomers of chemical compounds in a complex mixture. The method makes use of fast field cycling such that the spin evolution takes place under ZULF conditions, whereas signal detection is performed in a high-field NMR spectrometer. This method has excellent sensitivity, also allowing easy assignment of ZULF-NMR spectra to specific analytes in the mixture. We demonstrate that the spectral information is the same as that given by ZULF-NMR, which makes the method suitable for creating a library of ZULF-NMR spectra of various compounds and their isotopomers. The results of the field-cycling experiments can be presented in a convenient way as 2D-NMR spectra with the direct dimension giving the high-field 13C-NMR spectrum (carrying the chemical-shift information) and the indirect dimension giving the ZULF-NMR spectrum (containing information about proton-carbon J-couplings). Hence, the method can be seen as a variant of heteronuclear J-resolved spectroscopy, one of the first 2D-NMR techniques.
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Signal amplification by reversible exchange (SABRE) boosts NMR signals of various nuclei enabling new applications spanning from magnetic resonance imaging to analytical chemistry and fundamental physics. SABRE is especially well positioned for continuous generation of enhanced magnetization on a large scale; however, several challenges need to be addressed for accomplishing this goal. Specifically, SABRE requires (i) a specialized catalyst capable of reversible H2 activation and (ii) physical transfer of the sample from the point of magnetization generation to the point of detection (e.g., a high-field or a benchtop nuclear magnetic resonance [NMR] spectrometer). Moreover, (iii) continuous parahydrogen bubbling accelerates solvent (e.g., methanol) evaporation, thereby limiting the experimental window to tens of minutes per sample. In this work, we demonstrate a strategy to rapidly generate the best-to-date precatalyst (a compound that is chemically modified in the course of the reaction to yield the catalyst) for SABRE, [Ir(IMes)(COD)Cl] (IMes = 1,3-bis-[2,4,6-trimethylphenyl]-imidazol-2-ylidene; COD = cyclooctadiene) via a highly accessible synthesis. Second, we measure hyperpolarized samples using a home-built zero-field NMR spectrometer and study the field dependence of hyperpolarization directly in the detection apparatus, eliminating the need to physically move the sample during the experiment. Finally, we prolong the measurement time and reduce evaporation by presaturating parahydrogen with the solvent vapor before bubbling into the sample. These advancements extend opportunities for exploring SABRE hyperpolarization by researchers from various fields and pave the way to producing large quantities of hyperpolarized material for long-lasting detection of SABRE-derived nuclear magnetization.
Asunto(s)
Imagen por Resonancia Magnética , Catálisis , Espectroscopía de Resonancia MagnéticaRESUMEN
Iron-sulfur clusters are protein cofactors with an ancient evolutionary origin. These clusters are best known for their roles in redox proteins such as ferredoxins, but some iron-sulfur clusters have nonredox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize. Here we report a structural and functional characterization of dihydroxyacid dehydratase (DHAD) from Mycobacterium tuberculosis (Mtb), an essential enzyme in the biosynthesis of branched-chain amino acids. Conducting this analysis under fully anaerobic conditions, we solved the DHAD crystal structure, at 1.88 Å resolution, revealing a 2Fe-2S cluster in which one iron ligand is a potentially exchangeable water molecule or hydroxide. UV and EPR spectroscopy both suggested that the substrate binds directly to the cluster or very close to it. Kinetic analysis implicated two ionizable groups in the catalytic mechanism, which we postulate to be Ser-491 and the iron-bound water/hydroxide. Site-directed mutagenesis showed that Ser-491 is essential for activity, and substrate docking indicated that this residue is perfectly placed for proton abstraction. We found that a bound Mg2+ ion 6.5 Å from the 2Fe-2S cluster plays a key role in substrate binding. We also identified a putative entry channel that enables access to the cluster and show that Mtb-DHAD is inhibited by a recently discovered herbicide, aspterric acid, that, given the essentiality of DHAD for Mtb survival, is a potential lead compound for the design of novel anti-TB drugs.
Asunto(s)
Aminoácidos de Cadena Ramificada/biosíntesis , Hidroliasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Mycobacterium tuberculosis/química , Aminoácidos de Cadena Ramificada/química , Sitios de Unión , Hidroliasas/química , Proteínas Hierro-Azufre/química , Modelos Moleculares , Conformación Molecular , Mycobacterium tuberculosis/metabolismoRESUMEN
We demonstrate that heterogeneous/biphasic chemical reactions can be monitored with high spectroscopic resolution using zero-field nuclear magnetic resonance spectroscopy. This is possible because magnetic susceptibility broadening is negligible at ultralow magnetic fields. We show the two-step hydrogenation of dimethyl acetylenedicarboxylate with para-enriched hydrogen gas in conventional glass NMR tubes, as well as in a titanium tube. The low frequency zero-field NMR signals ensure that there is no significant signal attenuation arising from shielding by the electrically conductive sample container. This method paves the way for inâ situ monitoring of reactions in complex heterogeneous multiphase systems and in reactors made of conductive materials while maintaining resolution and chemical specificity.
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We report the results of a search for axionlike dark matter using nuclear magnetic resonance (NMR) techniques. This search is part of the multifaceted Cosmic Axion Spin Precession Experiment program. In order to distinguish axionlike dark matter from magnetic fields, we employ a comagnetometry scheme measuring ultralow-field NMR signals involving two different nuclei (^{13}C and ^{1}H) in a liquid-state sample of acetonitrile-2-^{13}C (^{13}CH_{3}CN). No axionlike dark matter signal was detected above the background. This result constrains the parameter space describing the coupling of the gradient of the axionlike dark matter field to nucleons to be g_{aNN}<6×10^{-5} GeV^{-1} (95% confidence level) for particle masses ranging from 10^{-22} eV to 1.3×10^{-17} eV, improving over previous laboratory limits for masses below 10^{-21} eV. The result also constrains the coupling of nuclear spins to the gradient of the square of the axionlike dark matter field, improving over astrophysical limits by orders of magnitude over the entire range of particle masses probed.
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We show that in a spin system of two magnetically inequivalent protons coupled to a heteronucleus such as 13C, an adiabatic magnetic field sweep, passing through zero field, transfers the proton singlet order into magnetization of the coupled heteronucleus. This effect is potentially useful in parahydrogen-enhanced nuclear magnetic resonance and is demonstrated on singlet-hyperpolarized [1-13C]maleic acid, which is prepared via the reaction between [1-13C]acetylene dicarboxylic acid and para-enriched hydrogen gas. The magnetic field sweeps are of microtesla amplitudes and have durations on the order of seconds. We show a polarization enhancement by a factor of 104 in the 13C spectra of [1-13C]maleic acid in a 1.4 T magnetic field.
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The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, ß-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.
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Proteínas Bacterianas/química , Mycobacterium smegmatis/enzimología , Treonina Deshidratasa/química , Regulación Alostérica/fisiología , Proteínas Bacterianas/metabolismo , Catálisis , Cinética , Especificidad por Sustrato/fisiología , Treonina Deshidratasa/metabolismoRESUMEN
Atomic comagnetometers are used in searches for anomalous spin-dependent interactions. Magnetic field gradients are one of the major sources of systematic errors in such experiments. Here we describe a comagnetometer based on the nuclear spins within an ensemble of identical molecules. The dependence of the measured spin-precession frequency ratio on the first-order magnetic field gradient is suppressed by over an order of magnitude compared to a comagnetometer based on overlapping ensembles of different molecules. Our single-species comagnetometer is capable of measuring the hypothetical spin-dependent gravitational energy of nuclei at the 10^{-17} eV level, comparable to the most stringent existing constraints. Combined with techniques for enhancing the signal such as parahydrogen-induced polarization, this method of comagnetometry offers the potential to improve constraints on spin-gravity coupling of nucleons by several orders of magnitude.
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l-Aspartate oxidase, encoded by the nadB gene, is the first enzyme in the de novo synthesis of NAD+ in bacteria. This FAD-dependent enzyme catalyzes the oxidation of l-aspartate to generate iminoaspartate and reduced flavin. Distinct from most amino acid oxidases, it can use either molecular oxygen or fumarate to reoxidize the reduced enzyme. Sequence alignments and the three-dimensional crystal structure have revealed that the overall fold and catalytic residues of NadB closely resemble those of the succinate dehydrogenase/fumarate reductase family rather than those of the prototypical d-amino acid oxidases. This suggests that the enzyme can catalyze amino acid oxidation via typical amino acid oxidase chemistry, involving the removal of protons from the α-amino group and the transfer of the hydride from C2, or potentially deprotonation at C3 followed by transfer of the hydride from C2, similar to chemistry occurring during succinate oxidation. We have investigated this potential mechanistic ambiguity using a combination of primary, solvent, and multiple deuterium kinetic isotope effects in steady state experiments. Our results indicate that the chemistry is similar to that of typical amino acid oxidases in which the transfer of the hydride from C2 of l-aspartate to FAD is rate-limiting and occurs in a concerted manner with respect to deprotonation of the α-amine. Together with previous kinetic and structural data, we propose that NadB has structurally evolved from succinate dehydrogenase/fumarate reductase-type enzymes to gain the new functionality of oxidizing amino acids while retaining the ability to reduce fumarate.
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Aminoácido Oxidorreductasas/metabolismo , Ácido Aspártico/metabolismo , Coenzimas/metabolismo , Escherichia coli K12/enzimología , Proteínas de Escherichia coli/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Modelos Moleculares , Algoritmos , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/genética , Animales , Ácido Aspártico/química , Sitios de Unión , Biocatálisis , Dominio Catalítico , Coenzimas/química , Medición de Intercambio de Deuterio , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleótido/química , Concentración de Iones de Hidrógeno , Cinética , Malato Deshidrogenasa/metabolismo , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sus scrofaRESUMEN
The eight enzymes responsible for the biosynthesis of the three branched-chain amino acids (l-isoleucine, l-leucine, and l-valine) were identified decades ago using classical genetic approaches based on amino acid auxotrophy. This review will highlight the recent progress in the determination of the three-dimensional structures of these enzymes, their chemical mechanisms, and insights into their suitability as targets for the development of antibacterial agents. Given the enormous rise in bacterial drug resistance to every major class of antibacterial compound, there is a clear and present need for the identification of new antibacterial compounds with nonoverlapping targets to currently used antibacterials that target cell wall, protein, mRNA, and DNA synthesis.
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Aminoácidos de Cadena Ramificada/biosíntesis , Antibacterianos/farmacología , Enzimas/efectos de los fármacos , Antibacterianos/química , Enzimas/química , Conformación MolecularRESUMEN
Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.