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ABSTRACT: Intermediate species during an anionic polymerization of styrene are observed by hyperpolarized nuclear magnetic resonance (NMR). Dissolution dynamic nuclear polarization (DNP) of monomers provides a sufficient signal-to-noise ratio to detect 13C NMR signals in real time while the reaction progresses. Due to a large chemical shift dispersion, 13C is well suited to distinguish and characterize the chemical species that arise during the reaction. At the same time, incorporation of hyperpolarized small-molecule monomers represents a unique way of generating polymer that exhibits a transient signal enhancement at the active site. This strategy is applicable despite the rapid spin-lattice relaxation in the final product that would exacerbate the direct hyperpolarization of the macromolecule using dissolution DNP. In the study of polymerization reactions, the real-time measurement gives access to both mechanistic and kinetic information. Further, it does not require stable isotope labeling of the molecules of interest. These capabilities are orthogonal to currently established methods that separate synthesis and analysis into two steps, making dissolution DNP an attractive method for the study of polymerization reactions.
Journal of the American Chemical Society 03/2013; · 9.91 Impact Factor
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ABSTRACT: Fluorine NMR spectroscopy is widely used for detection of protein-ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug molecule to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiologically high protein concentrations and large ligand:protein ratios. An enhancement in the (19)F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concentrations of fluorinated small molecules. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR analysis, dissociation constants are determined. However, the ability to use a low ligand concentration permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and additional information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery.
Journal of the American Chemical Society 09/2012; 134(42):17448-51. · 9.91 Impact Factor
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ABSTRACT: Short, secondary-structure-containing peptides are suitable models for the study of protein folding due to their relative simplicity. Here, we investigate thermal denaturation of the tryptophan zipper peptide, trpzip4, a peptide that forms a β-hairpin in solution. In order to monitor the thermal denaturation of peptides or small proteins, chemical shift values of H(α) or H(N) may be used. However, various factors other than secondary structure can influence chemical shift values, such as side-chain orientation of nearby aromatic residues. Nuclear Overhauser effect (NOE) intensity from backbone interproton cross peaks is an alternative way to study thermal denaturation, as long as various factors that give rise to a change in NOE intensity upon changing the temperature are considered. As a relative indicator for denaturation, we define a cutoff temperature, where half of the initial NOE intensity is lost for each backbone interproton cross peak. For trpzip4, this cutoff temperature is highest for residues in the central part of the structure and lowest for residues near the termini. These observations support the notion that the structure of the trpzip4 peptide is stabilized by a hydrophobic cluster formed by tryptophan residues located in the central region of the β-hairpin.
The Journal of Physical Chemistry B 12/2011; 115(51):15355-61. · 3.70 Impact Factor
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ABSTRACT: Hyperpolarization of nuclear spins through techniques such as dynamic nuclear polarization (DNP) can greatly increase the signal-to-noise ratio in NMR measurements, thus eliminating the need for signal averaging. This enables the study of many dynamic processes which would otherwise not be amenable to study by NMR spectroscopy. A report of solid- to liquid-state DNP of a short peptide, bacitracin A, as well as of a full-length protein, L23, is presented here. The polypeptides are hyperpolarized at low temperature and dissolved for NMR signal acquisition in the liquid state in mixtures of organic solvent and water. Signal enhancements of 300-2000 are obtained in partially deuterated polypeptide when hyperpolarized on (13)C and of 30-180 when hyperpolarized on (1)H. A simulated spectrum is used to identify different resonances in the hyperpolarized (13)C spectra, and the relation between observed signal enhancement for various groups in the protein and relaxation parameters measured from the hyperpolarized samples is discussed. Thus far, solid- to liquid-state DNP has been used in conjunction with small molecules. The results presented here, however, demonstrate the feasibility of hyperpolarizing larger proteins, with potential applications toward the study of protein folding or macromolecular interactions.
Analytical Chemistry 06/2011; 83(15):6054-9. · 5.86 Impact Factor
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ABSTRACT: A combined simulation and experimental study was performed to investigate how methanol affects the structure of a model peptide BBA5. BBA5 forms a stable β-hairpin-α-helix structure in aqueous solutions. Molecular dynamics simulations were performed in water and methanol/water solutions using all-atom explicit models. NMR experiments were used to test the calculated results. The combined theoretical and experimental studies suggest that methanol strengthens the interactions between the polar backbone of the peptide and thus enhances the secondary structure formation; at the same time methanol weakens the hydrophobic interactions and results in an expansion of the hydrophobic core and an increase in gyration.
The Journal of Physical Chemistry B 05/2011; 115(20):6653-60. · 3.70 Impact Factor
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ABSTRACT: 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.
NMR in Biomedicine 03/2011; 24(8):1016-22. · 3.21 Impact Factor
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ABSTRACT: 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.
Journal of Magnetic Resonance 02/2011; 208(2):204-9. · 2.14 Impact Factor
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ABSTRACT: The two-stage model for membrane protein folding postulates that individual helices form first and are subsequently packed against each other. To probe the two-stage model, the structures of peptides representing individual transmembrane helices of the disulfide bond forming protein B have been studied in trifluoroethanol solution as well as in detergent micelles using nuclear magnetic resonance (NMR) and circular dichroism spectroscopy. In TFE solution, peptides showed well-defined α-helical structures. Peptide structures in TFE were compared to the structures of full-length protein obtained by X-ray crystallography and NMR. The extent of α-helical secondary structure coincided well, lending support for the two-stage model for membrane protein folding. However, the conformation of some amino acid side chains differs between the structures of peptide and full-length protein. In micellar solution, the peptides also adopted a helical structure, albeit of reduced definition. Using measurements of paramagnetic relaxation enhancement, peptides were confirmed to be embedded in micelles. These observations may indicate that in the native protein, tertiary interactions additionally stabilize the secondary structure of the individual transmembrane helices.
Proteins Structure Function and Bioinformatics 01/2011; 79(5):1365-75. · 3.39 Impact Factor
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ABSTRACT: Emerging techniques for hyperpolarization of nuclear spins, foremost dynamic nuclear polarization (DNP), lend unprecedented sensitivity to nuclear magnetic resonance spectroscopy. Sufficient signal can be obtained from a single scan, and reactions even far from equilibrium can be studied in real-time. When following the progress of a reaction by nuclear magnetic resonance, however, spin relaxation occurs concomitantly with the reaction to alter resonance line intensities. Here, we present a model for accounting for spin-relaxation in such reactions studied by hyperpolarized NMR. The model takes into account auto- and cross-relaxation in dipole-dipole coupled spin systems and is therefore applicable to NMR of hyperpolarized protons, the most abundant NMR-active nuclei. Applied to the Diels-Alder reaction of 1,4-dipheneylbutadiene (DPBD) with 4-phenyl-1,2,4-triazole-3,5-dione (PTD), reaction rates could be obtained accurately and reproducibly. Additional parameters available from the same experiment include relaxation rates of the reaction product, which may yield further information about the molecular properties of the product. The method presented is also compatible with an experiment where a single spin in the reactant is labeled in its spin-state by a selective radio frequency pulse for subsequent tracking through the reaction, allowing the unambiguous identification of its position in the product molecule. In this case, the chemical shift specificity of high-resolution NMR can allow for the simultaneous determination of reaction rates and mechanistic information in one experiment.
Analytical Chemistry 10/2010; · 5.86 Impact Factor
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ABSTRACT: 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.
Organic & Biomolecular Chemistry 08/2010; 8(15):3361-5. · 3.70 Impact Factor
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ABSTRACT: 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.
Physical Chemistry Chemical Physics 06/2010; 12(22):5766-70. · 3.57 Impact Factor
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ABSTRACT: Nuclear magnetic resonance (NMR) poses significant challenges for teaching in the context of an undergraduate laboratory, foremost because of high equipment cost. Current off-the-shelf data-acquisition hardware, however, is sufficiently powerful to constitute the core of a fully digital NMR spectrometer operating at the earth’s field. We present an NMR experiment that we have developed on this basis for an upper-division laboratory in physical chemistry. Students utilize a spectrometer that is controlled by a program written in LabView to explore the fundamentals of NMR alongside modern techniques of digital data acquisition. An advantage of this approach is that all of the hardware and software components used in the experiment are transparent to the user, which fosters inquiry-based discovery of materials beyond the basic experiments described in the student manual.
Journal of chemical education 05/2010; 87(7):2010. · 0.74 Impact Factor
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ABSTRACT: 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.
Analytical Chemistry 05/2009; 81(11):4543-7. · 5.86 Impact Factor
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ABSTRACT: A scheme capable of acquiring heteronuclear 2D NMR spectra of hyperpolarized sample is described. Hyperpolarization, the preparation of nuclear spins in a polarized state far from thermal equilibrium, can increase the NMR signal by several orders of magnitude. It presents opportunities to apply NMR spectroscopy to dilute samples that would otherwise yield insufficient signal. However, conventional 2D NMR spectroscopy, which is commonly applied for the determination of molecular structure, relies on the recovery of the initial polarization after each transient. For this reason, it cannot be applied directly to a sample that has been hyperpolarized once. With appropriately modified pulse schemes, two-dimensional NMR spectra an however be acquired sequentially by utilizing a small portion of the hyperpolarized signal in every scan, while keeping the remaining polarization for future scans. We present heteronuclear multi-quantum spectra of single hyperpolarized samples using this technique, and discuss different options for distributing the polarization among different scans. This robust method takes full advantage of Fourier NMR to resolve overlapping chemical shifts, and may prove particularly useful for the structural elucidation of compounds in mass-limited samples.
Journal of Magnetic Resonance 05/2009; 199(2):159-65. · 2.14 Impact Factor
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ABSTRACT: A scheme capable of acquiring heteronuclear 2D NMR spectra of hyperpolarized sample is described. Hyperpolarization, the preparation of nuclear spins in a polarized state far from thermal equilibrium, can increase the NMR signal by several orders of magnitude. It presents opportunities to apply NMR spectroscopy to dilute samples that would otherwise yield insufficient signal. However, conventional 2D NMR spectroscopy, which is commonly applied for the determination of molecular structure, relies on the recovery of the initial polarization after each transient. For this reason, it cannot be applied directly to a sample that has been hyperpolarized once. With appropriately modified pulse schemes, two-dimensional NMR spectra an however be acquired sequentially by utilizing a small portion of the hyperpolarized signal in every scan, while keeping the remaining polarization for future scans. We present heteronuclear multi-quantum spectra of single hyperpolarized samples using this technique, and discuss different options for distributing the polarization among different scans. This robust method takes full advantage of Fourier NMR to resolve overlapping chemical shifts, and may prove particularly useful for the structural elucidation of compounds in mass-limited samples.
Journal of Magnetic Resonance 04/2009; 199(2):159-165. · 2.14 Impact Factor
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ABSTRACT: 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.
Analytical Chemistry 08/2008; 80(15):5794-8. · 5.86 Impact Factor
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Angewandte Chemie International Edition 01/2008; 47(28):5235-7. · 13.45 Impact Factor
