Modern Nuclear Magnetic Resonance Research Articles

"Rules of Engagement" of Protein-Glycoconjugate Interactions: A Molecular View Achievable by using NMR Spectroscopy and Molecular Modeling.

Understanding the dynamics of protein–ligand interactions, which lie at the heart of host–pathogen recognition, represents a crucial step to clarify the molecular determinants implicated in binding events, as well as to optimize the design of new molecules with therapeutic aims. Over the last decade, advances in complementary biophysical and spectroscopic methods permitted us to deeply dissect the fine structural details of biologically relevant molecular recognition processes with high resolution. This Review focuses on the development and use of modern nuclear magnetic resonance (NMR) techniques to dissect binding events. These spectroscopic methods, complementing X‐ray crystallography and molecular modeling methodologies, will be taken into account as indispensable tools to provide a complete picture of protein–glycoconjugate binding mechanisms related to biomedicine applications against infectious diseases.

Network Former Mixing (NFM) Effects in Ion-Conducting Glasses - Structure/Property Correlations Studied by Modern Solid-State NMR Techniques

Glassy solid electrolytes are important integral components for all-solid-state devices for energy storage and conversion. The use of multiple network formers is an important part of their design strategy for specific applications. In many glass systems the interaction between the different network formers results in strongly non-linear variations in physical properties (network former mixing (NFM) effects), requiring a detailed understanding on a structural basis.The issues to be addressed involve both the structural organization and connectivities within the framework, the local environments and spatial distributions of the mobile ions, and the dynamical aspects of ion transport, to be discussed in relation to possible phase separation or nano-segregation effects. Besides Raman and X-ray photoelectron spectroscopies, solid state nuclear magnetic resonance (NMR) methods are particularly useful for providing detailed answers to such issues. The present review introduces the basic principles of modern solid state NMR methods and their applications to glass structure, with a particular focus on the characterization of network-former mixing effects in the most common lithium and sodium conducting oxide and chalcogenide glass systems. Based on the current state of the literature reviewed in the present work, some emerging general principles governing structure/property correlations are identified, to be tested by further experimenteation in the future.

Multidimensional J-driven NMR correlations by single-scan offset-encoded recoupling

Two-dimensional (2D) correlations between bonded heteroatoms, lie at the cornerstone of many uses given to contemporary nuclear magnetic resonance (NMR). Improving the efficiency with which these correlations are established is an important topic in modern NMR, with potential applications in rapid chemical analysis and dynamic biophysical studies. Alternatives have been developed over the last decade to speed up these experiments, based among others on reducing the number of data points that need to be sampled, and/or shortening the inter-scan delays. Approaches have also been proposed to forfeit multi-scan schemes altogether, and complete full 2D correlations in a single shot. Here we explore and discuss a new alternative enabling the collection of such very fast – in principle, single-scan – acquisitions of 2D heteronuclear correlations among bonded species, which operates on the basis of a partial reintroduction of J couplings. Similar approaches had been proposed in the past based on collecting coupled spectra for arrays of off-resonance decoupling values; the proposal that is here introduced operates on the basis of suitably incorporating frequency-swept pulses, into spin-echo sequences. Thanks to the offset-dependent amplitude modulations of the in- and anti-phase components that such sequences impart, chemical shifts of coupled but otherwise unobserved nuclear species, can be extracted from the relative intensities and phases of J-coupled multiplets observed in one-dimensional acquisitions. A description of the steps needed to implement this rapid acquisition approach in a quantitative fashion, as well as applications of the ensuing sequences, are presented.

Spatially resolved spectroscopy using tapered stripline NMR

Magnetic field B0 gradients are essential in modern Nuclear Magnetic Resonance spectroscopy and imaging. Although RF/B1 gradients can be used to fulfill a similar role, this is not used in common practice because of practical limitations in the design of B1 gradient coils. Here we present a new method to create B1 gradients using stripline RF coils. The conductor-width of a stripline NMR chip and the strength of its radiofrequency field are correlated, so a stripline chip can be tapered to produce any arbitrary shaped B1 field gradient. Here we show the characterization of this tapered stripline configuration and demonstrate three applications: magnetic resonance imaging on samples with nL–μL volumes, reaction monitoring of fast chemical reactions (10−2–101s) and the compensation of B0 field gradients to obtain high-resolution spectra in inhomogeneous magnetic fields.

A peripheral component interconnect express-based scalable and highly integrated pulsed spectrometer for solution state dynamic nuclear polarization

High sensitivity, high data rates, fast pulses, and accurate synchronization all represent challenges for modern nuclear magnetic resonance spectrometers, which make any expansion or adaptation of these devices to new techniques and experiments difficult. Here, we present a Peripheral Component Interconnect Express (PCIe)-based highly integrated distributed digital architecture pulsed spectrometer that is implemented with electron and nucleus double resonances and is scalable specifically for broad dynamic nuclear polarization (DNP) enhancement applications, including DNP-magnetic resonance spectroscopy/imaging (DNP-MRS/MRI). The distributed modularized architecture can implement more transceiver channels flexibly to meet a variety of MRS/MRI instrumentation needs. The proposed PCIe bus with high data rates can significantly improve data transmission efficiency and communication reliability and allow precise control of pulse sequences. An external high speed double data rate memory chip is used to store acquired data and pulse sequence elements, which greatly accelerates the execution of the pulse sequence, reduces the TR (time of repetition) interval, and improves the accuracy of TR in imaging sequences. Using clock phase-shift technology, we can produce digital pulses accurately with high timing resolution of 1 ns and narrow widths of 4 ns to control the microwave pulses required by pulsed DNP and ensure overall system synchronization. The proposed spectrometer is proved to be both feasible and reliable by observation of a maximum signal enhancement factor of approximately -170 for (1)H, and a high quality water image was successfully obtained by DNP-enhanced spin-echo (1)H MRI at 0.35 T.

The complex isotopologue space of glucose as a framework for the study of human intermediary metabolism

The positional distributions of stable isotopes in metabolites provide specific fingerprints of the pathways and fluxes that have occurred in the organisms under study. In particular, modern nuclear magnetic resonance (NMR) spectroscopy enables the detailed assignment of isotope patterns in natural products, for example, in metabolites obtained from labelling experiments using 13C-enriched precursors, such as glucose, acetate or CO2. In this study, the transient 13C-isotopologue composition of blood glucose from an adult human volunteer after intravenous supply of [U-13C6]glucose was determined by high-resolution 13C NMR spectroscopy. The non-linear progression curves displaying the relative amounts of eight 13C-glucose isotopologues reflected the contributions of glucose metabolism by glycolytic cycling, the pentose phosphate pathway and anaplerotic reactions involving the citric acid cycle. The pilot study suggests that the experimental setting can be useful in analysing under non-invasive conditions the impact of physiological and pharmacological constraints on glucose turnover in humans.

Nuclear magnetic resonance in the structural elucidation of natural products.

From its modest beginnings in the 1950s, nuclear magnetic resonance (NMR) spectroscopy has become the premier analytical tool for the determination of structure of organic natural products. Structural elucidation efforts were originally limited to proton NMR and typically required both relatively large quantities of material and considerable time. However, modern NMR spectrometers, with an array of one- and two-dimensional experiments, permit the structures of complex organic molecules to be determined, often in a day, using less than 1 mg of sample. This chapter will prepare natural product chemists to employ modern NMR techniques effectively in the determination of molecular structures. It focuses on the rapid determination of whether an isolated compound is known or new, the information content of various two-dimensional and selective one-dimensional NMR experiments, the use of these experiments in combination and avoiding or overcoming common pitfalls in determining molecular structures, the selection of optimum acquisition parameters and data processing methods and parameters, and the use of computer-assisted structure elucidation.

The linker between the dimerization and catalytic domains of the CheA histidine kinase propagates changes in structure and dynamics that are important for enzymatic activity.

The histidine kinase, CheA, couples environmental stimuli to changes in bacterial swimming behavior, converting a sensory signal to a chemical signal in the cytosol via autophosphorylation. The kinase activity is regulated in the platform of chemotaxis signaling complexes formed by CheW, chemoreceptors, and the regulatory domain of CheA. Our previous computational and mutational studies have revealed that two interdomain linkers play important roles in CheA’s enzymatic activity. Of the two linkers, one that connects the dimerization and ATP binding domains is essential for both basal autophosphorylation and activation of the kinase. However, the mechanistic role of this linker remains unclear, given that it is far from the autophosphorylation reaction center (the ATP binding site). Here we investigate how this interdomain linker is coupled to CheA’s enzymatic activity. Using modern nuclear magnetic resonance (NMR) techniques, we find that by interacting with the catalytic domain, the interdomain linker initiates long-range structural and dynamic changes directed toward the catalytic center of the autophosphorylation reaction. Subsequent biochemical assays define the functional relevance of these NMR-based observations. These findings extend our understanding of the chemotaxis signal transduction pathway.

Capturing Brain Metrics of Neuropathic Pain Using Nuclear Magnetic Resonance

SUMMARYNeuropathic pain is typically caused by a lesion or dysfunction in the nervous system that results in both negative (i.e., reduced sensitivity) and positive symptoms (i.e., paraesthesia, spontaneous ongoing pain with shooting, electric shock-like sensations and abnormal responses to evoked pain). Intriguingly, chronic pain disorders manifest profound alterations in brain structure and function, and thus, modern nuclear magnetic resonance (NMR) techniques have allowed us to begin to dissect the complexities of how neuropathic pain affects the brain. NMR approaches can be used as an independent measure to improve our understanding of key changes in brain structure, function and chemistry in chronic neuropathic pain. Accordingly, NMR techniques provide neurobiological metrics that allow us to understand the neurobiological basis of chronic neuropathic pain. Additionally, although at an early stage, NMR methods can also be useful to define clinical metrics to predict chronification of neuropathic pain and responses to drugs. This article provides a review of NMR techniques and their capacity to study spontaneous pain and evoked pain, as well as structural, functional and neurochemical alterations that have repeatedly been associated with chronic neuropathic pain. Finally, the importance for quantifying disease state and treatment efficacy in neuropathic pain using NMR techniques is discussed.

Description of pulse NMR experiments by means of the product operator formalism

A simple technique based on the product operator formalism, which provides a complete and rigorous quantum-mechanical description of modern pulse nuclear magnetic resonance experiments is proposed. Calculation schemes for unambiguous assignment of chemical shifts in nuclear magnetic resonance spectra for two- and three-spin systems and for measuring spectral parameters needed for the structural analysis of molecules are presented.

Soil Inositol Phosphate Biochemistry: A Call to Link Radiotracer Applications with Spectroscopic Structural Analysis

Early work on the use of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy for the structural analysis of soil inositol phosphates and the use of radioisotope tracers to identify and measure soil inositol phosphate transformations is briefly described. Modern advances in the application of MS and NMR to the identification and quantification of soil inositol phosphate stereoisomers including advances in on‐line liquid chromatography (LC) coupled to MS and NMR in hyphenated LC–MS–NMR techniques are reviewed. Automated techniques used to measure 3H, 14C, 33P, and 32P with on‐line LC–NMR and LC–MS are described. Recommendations are made for the use of radioisotope‐labeled myo‐, D‐chiro‐, scyllo‐, and neo‐inositol and their phosphorylated forms in conjunction with the application of modern MS and NMR spectroscopic structural analysis in studies on inositol phosphate transformations in soils.

Unraveling the long-term stabilization mechanisms of organic materials in soils by physical fractionation and NMR spectroscopy

The fundamental mechanisms whereby organic inputs stabilize in soil are poorly resolved, which limits our current capacity to predict the dynamics of soil organic matter (OM) turnover and its influence on soil quality and functioning. Here we fractionated soil OM from long-term experimental field plots either unamended or amended with two organic materials of different quality (i.e., solid cattle manure and crop residues) for 44 years into five measurable and meaningful pools directly related to conceptual preservation mechanisms: dissolved OM, mineral-free particulate OM located outside aggregates (unprotected from decomposition), OM occluded within both macroaggregates and microaggregates (weakly and strongly protected by physical mechanisms, respectively), and OM intimately associated with soil mineral particles (protected by chemical mechanisms). Compared to the unamended soil, the application of cattle manure and crop residues increased total organic C content by 35 and 10%, respectively. Most of these increases (up to 60 and 72% for cattle manure and crop residues, respectively) were explained by the mineral-associated OM pool, followed by the intra-microaggregate OM fraction. In general, the distribution and dynamics of N content paralleled those of C content. As determined by a range of modern nuclear magnetic resonance (NMR) techniques, including 13C cross polarization magic angle spinning (MAS), 1H high resolution (HR)-MAS, and 1H13C heteronuclear single quantum coherence HR-MAS NMR, the mineral-associated OM fraction was found to be predominately of microbial origin, unlike free and intra-aggregate OM pools, which were dominated by plant structures at different stages of decomposition. As a whole, our results indicate that the main mechanism by which organic inputs are stabilized and OM accrues in soils is not the physical and chemical protection of undecayed or partially degraded organic structures, but the adsorption on mineral surfaces of microbial biomass and microbial by-products resulting from microbial growth, transformation, and degradation processes. It is possible that organic amendments increase more than previously thought the microbial populations of the soil, which live, thrive, and die in close association with the mineral surfaces. This mechanism appears to be enhanced with the addition of stable organic materials.

Paecilomide, a new acetylcholinesterase inhibitor from Paecilomyces lilacinus

Fungi are some of the most important organisms in the production of bioactive secondary metabolites. This success is related to the advances in biotechnology and also to the possibility of working with techniques such as the “OSMAC” (one strain-many compounds) to achieve different fungal secondary metabolites profiles upon modifying the culturing conditions. Using this approach, the fungal species Paecilomyces lilacinus was cultivated in potato dextrose broth under 14 different fermentative conditions by adding the bacterium Salmonella typhimurium to the growing medium in order to provide biotic stress. S. typhimurium was added alive or after inactivation by autoclave or microwave irradiation in different stages of fungal growth. Extracts were prepared by liquid–liquid extraction using ethyl acetate, a medium polarity solvent in order to avoid extracting culturing media components. Production of fatty acids of relevance for the pharmaceutical and food industries was enhanced by the modified fermentative conditions and they were identified and quantified. The extracts were evaluated for acetylcholinesterase inhibition and the more active extract (91±2.91% inhibition) was prepared in large scale. From this active P. lilacinus extract, a novel pyridone alkaloid, named Paecilomide, was isolated and its structure was elucidated by modern nuclear magnetic resonance techniques and mass spectrometric analyses. Paecilomide (1) was also evaluated for acetylcholinesterase inhibition, presenting 57.5±5.50% of acetylcholinesterase inhibition.

Nuclear Magnetic Resonance Spectroscopy and Its Key Role in Environmental Research

Nuclear magnetic resonance (NMR) is arguably the most powerful and versatile tool in modern science. It has the capability to solve complex structures and interactions in situ even in complex heterogeneous multiphase samples such as soil, plants, and tissues. NMR has vast potential in environmental research and can provide insight into a diverse range of environmental processes at the molecular level be it identifying the binding site in human blood for a specific contaminant or the compositional dynamics of soil with climate change. Modern NMR-based metabonomics is elucidating contaminant toxicity and toxic mode of action rapidly and at sub lethal concentrations. Combined modern NMR approaches provide a powerful framework to better understand carbon cycling and sustainable agriculture, as well as contaminant fate, bioavailability, toxicity, sequestration, and remediation.

Physical, chemical, and biochemical mechanisms of soil organic matter stabilization under conservation tillage systems: A central role for microbes and microbial by-products in C sequestration

Conservation tillage practices that entail no or reduced soil disturbance are known to help preserve or accumulate soil organic matter (OM). However, the underlying mechanisms especially at the molecular level are not well understood. In this study soil samples from 25-year-old experimental plots continuously cropped with barley (Hordeum vulgare L.) under no-tillage (NT) and chisel tillage (CT) were subjected to a new physical fractionation method to isolate dissolved OM, mineral-free particulate OM located outside aggregates (physically and chemically unprotected), OM occluded within both macroaggregates and microaggregates (weakly and strongly protected by physical mechanisms, respectively), and OM in intimate association with minerals (protected by chemical mechanisms). The whole soils and OM fractions were analyzed for organic C and N content and by modern nuclear magnetic resonance (NMR) techniques. The soil under NT stored 16% more organic C and 5% more N than the soil under CT. Compared to CT, NT increased free organic C content by 7%, intra-macroaggregate organic C content by 20%, intra-microaggregate organic C content by 63%, and mineral-associated organic C content by 16% and decreased dissolved organic C content by 11%. The mineral-associated OM pool accounted for 65% of the difference in total organic C content between NT and CT, whereas the intra-microaggregate OM only explained 18%, intra-macroaggregate OM 14%, and free OM 11%. The NMR experiments revealed that the free and intra-aggregate OM fractions were dominated by crop-derived materials at different stages of decomposition, whereas the mineral-associated OM pool was predominately of microbial origin. Overall, our results indicate that microbes and microbial by-products associated with mineral surfaces and likely physically protected by entrapment within very small microaggregates constitute the most important pool of OM stabilization and C sequestration in soils under NT. Most probably the slower macroaggregate turnover in NT relative to CT boosts not only the formation of microaggregates and thereby the physical protection of crop-derived particulate OM, but more importantly the interaction between mineral particles and microbial material that results in the formation of very stable organo-mineral complexes.

Solution Structure of a Complex of the Histidine Autokinase CheA with Its Substrate CheY

In the bacterial chemotaxis two-component signaling system, the histidine-containing phosphotransfer domain (the "P1" domain) of CheA receives a phosphoryl group from the catalytic domain (P4) of CheA and transfers it to the cognate response regulator (RR) CheY, which is docked by the P2 domain of CheA. Phosphorylated CheY then diffuses into the cytoplasm and interacts with the FliM moiety of the flagellar motors, thereby modulating the direction of flagellar rotation. Structures of various histidine phosphotransfer domains (HPt) complexed with their cognate RR domains have been reported. Unlike the Escherichia coli chemotaxis system, however, these systems lack the additional domains dedicated to binding to the response regulators, and the interaction of an HPt domain with an RR domain in the presence of such a domain has not been examined on a structural basis. In this study, we used modern nuclear magnetic resonance techniques to construct a model for the interaction of the E. coli CheA P1 domain (HPt) and CheY (RR) in the presence of the CheY-binding domain, P2. Our results indicate that the presence of P2 may lead to a slightly different relative orientation of the HPt and RR domains versus those seen in such complex structures previously reported.

The use of stable isotope labelling for the analytical chemistry of drugs

This perspective reviews the potential for stable isotope labelling to examine the metabolic transformations of drugs. The increased sensitivity and widespread availability of modern nuclear magnetic resonance (NMR) and high-resolution mass spectrometers will increase the application of stable isotopes to study drug metabolism. Creating mass doublets by mixing a natural isotopic abundance compound with a labelled isotopomer and applying stable isotope filtering to high resolution mass spectrometry allows one to rapidly identify drug metabolites in very complex samples, such as blood or urine. Applying this approach to drug metabolism will require a significant synthesis effort. The relatively small number of (13) C, (15) N, or (17,18) O-labelled precursors exacerbates this problem, making the synthesis of the labelled drug often more difficult than that of the parent compound. We have developed new strategies for stable isotope labelling of complex molecules based on the rich chemistry of [(13) C]methyl phenyl sulfide, where the phenylthio group acts as a stable, non-volatile carrier for the valuable (13) C-label. For example we have used [(13) C]methyl phenyl sulfide to prepare the three possible (13) C-isotopomers ([1-(13) C]-, [2-(13) C]-, [1,2-(13) C(2) ]) of the two carbon precursors, ethyl 2-(phenylthio) acetate and ethyl N,N-dimethyl oxamate. In each case, these two-carbon labelling precursors are asymmetric and the differential reactivity of the carbons allows for either/or (13) C-labelling in the products. We demonstrate the utility of these two carbon precursors in the synthesis of aromatic ring-labelled N-(4-hydroxyphenyl)acetamide (acetaminophen or paracetamol).

Interaction of a goose-type lysozyme with chitin oligosaccharides as determined by NMR spectroscopy

The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [(GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 × 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 × 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (ΔH = -6.1 kcal/mol and TΔS = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.

A Novel Method for the Investigation of Liquid/Liquid Distribution Coefficients and Interface Permeabilities Applied to the Water-Octanol-Drug System

In this work a new, accurate and convenient technique for the measurement of distribution coefficients and membrane permeabilities based on nuclear magnetic resonance (NMR) is described. This method is a novel implementation of localized NMR spectroscopy and enables the simultaneous analysis of the drug content in the octanol and in the water phase without separation. For validation of the method, the distribution coefficients at pH = 7.4 of four active pharmaceutical ingredients (APIs), namely ibuprofen, ketoprofen, nadolol, and paracetamol (acetaminophen), were determined using a classical approach. These results were compared to the NMR experiments which are described in this work. For all substances, the respective distribution coefficients found with the two techniques coincided very well. Furthermore, the NMR experiments make it possible to follow the distribution of the drug between the phases as a function of position and time. Our results show that the technique, which is available on any modern NMR spectrometer, is well suited to the measurement of distribution coefficients. The experiments present also new insight into the dynamics of the water-octanol interface itself and permit measurement of the interface permeability.

ChemInform Abstract: Application of 119Sn NMR Parameters

Publisher Summary This chapter reviews that the impact of 119Sn nuclear magnetic resonance (NMR) parameters, determined either by 119Sn NMR or in the NMR spectra of other nuclei, on tin chemistry is remarkable. Nowadays, tin chemical shifts coupling constants and nuclear spin relaxation, with respect to the physical principles, is growing. At the same time, the application of more or less sophisticated NMR techniques enables one to monitor the reactions, identify the unstable intermediates, discover their relevance for mechanistic implications, assign the structural features, investigate the intra- and intermolecular dynamic processes, or to compare the structures in the solid and liquid state. 119Sn NMR parameters, in addition to those of the more common nuclei, for example, 1H, 13C, or 31P, add to the attempts at a more complete description of the bonding situation. The chapter suggests that it must be emphasized that the great potential of modern NMR spectrometers can be exploited by a multinuclear approach to obtain a large self-consistent set of NMR data. This also includes the isotope induced chemical shifts and long-range coupling constants that have been neglected in the past.