An efficient use of the WATERGATE W5 sequence for observing a ligand binding with a protein receptor

An efficient pulse sequence for observing a ligand binding with a receptor has been developed by incorporating the WATERGATE W5 sequence. In the conventional water ligand observed via gradient spectroscopy (WaterLOGSY) techniques, the water resonance is selectively excited using, e.g. the double-pulsed field gradient spin-echo (DPFGSE) sequence at the initial portion of pulse sequence. In the current version, the modified WATERGATE W5 sequence is incorporated at the initial portion of the pulse sequence, and the resonance at the water frequency can be selectively reserved by the modified WATERGATE W5 sequence. The efficiency of ligand-observed NMR screening techniques has been demonstrated using the human serum albumin (HSA)-tryptophan complex.     

Improvement of double pulsed field gradient spin‐echo ROESY experiments for identification of bound waters

A novel pulse sequence incorporating the double pulsed field gradient spin‐echo (DPFGSE) and the gradient‐tailored excitation WATERGATE techniques is presented that has particular use for identifying bound waters in ¹⁵N‐labeled macromolecules. This sequence, DPFGSE–ROESY–HSQC, affords greater spectral sensitivity than the DPFGSE–ROESY–HMQC experiment which was previously presented and is consequently useful for rapidly obtaining reliable information for characterizing macromolecular bound water molecules. A significant enhancement in the sensitivity is achieved by using the gradient‐tailored excitation WATERGATE sequence in the reverse INEPT step as it allows the use of much higher receiver gains. Since coherence selection is not used, the sequence has improved sensitivity together with less spectral artifacts. The advantage of this pulse sequence is illustrated using ¹⁵N‐labeled ribonuclease T₁. Copyright © 2003 John Wiley & Sons, Ltd.     

Broadband WET: a novel technique for quantitative characterization of minor components in foods

NMR analysis of foods frequently suffers from a problem of dynamic range, which limits the detection of minor components due to the huge signals of water and major components such as sugars. In the present study, we propose a new method named as ‘broadband WET’. This pulse scheme was applied to persimmon fruit juice for saturating the resonances of water and sugars, which covered a broad bandwidth. In comparison with the conventional solvent suppression methods such as WET and DPFGSE‐WATERGATE, it was shown that broadband WET provided highly selective suppression of resonances covering an extensive bandwidth and quantitative signals of minor components without distortion. The proposed method is suitable to detect quantitative signals of the minor components with a high sensitivity. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of the self‐diffusion of poly(ethylene glycol)s in poly(vinyl alcohol) aqueous systems

We have measured the self‐diffusion coefficients of a series of oligo‐ and poly(ethylene glycol)s with molecular weights ranging from 150 to 10,000, in aqueous solutions and gels of poly(vinyl alcohol) (PVA), using the pulsed‐gradient spin‐echo NMR techniques. The PVA concentrations varied from 0 to 0.38 g/mL which ranged from dilute solutions to polymer gels. Effects of the diffusant size and polymer concentration on the self‐diffusion coefficients have been investigated. The temperature dependence of the self‐diffusion coefficients has also been studied for poly(ethylene glycol)s with molecular weights of 600 and 2,000. Several theoretical models based on different physical concepts are used to fit the experimental data. The suitability of these models in the interpretation of the self‐diffusion data is discussed. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 2396–2403, 1999     

Estimation of Self‐Diffusion Coefficients of Small Penetrants in Semicrystalline Polymers Using Single‐Sided NMR

A simple and fast way to measure proton self‐diffusion coefficients of small penetrant molecules in semicrystalline polymers is introduced. The approach takes advantage of the strong static gradient of a mobile single‐sided NMR sensor and it is demonstrated on PE samples with varying degrees of crystallinity fully saturated in either toluene or n‐hexane. The self‐diffusion coefficients were measured using the gradient stimulated echo sequence appended with a CPMG. It is also shown for the first time, with demonstration on PE plates several millimeter thick with different aging histories, that one‐dimensional profiles of self‐diffusion coefficients as a function of depth can be easily obtained.     

Using perdeuterated surfactant micelles to resolve mixture components in diffusion‐ordered NMR spectroscopy

Diffusion‐ordered NMR spectroscopy resolves mixture components on the basis of differences in their respective diffusion coefficients or molecular sizes. However, when components have near‐identical diffusion coefficients, they are not resolved in the diffusion dimension of a diffusion‐ordered spectroscopy (DOSY) spectrum. Adding surfactant micelles to these mixtures has been shown to enhance resolution when the component molecules interact differentially with the micelles. This approach is similar to that used in electrokinetic chromatography (EKC) where modifiers like micelles or polymers are used to enhance the separation of mixture components. In this study, perdeuterated surfactants are added to analyte mixtures studied with the DOSY technique. Since no micelle resonances appear in the mixture spectra, the difficulty associated with performing biexponential analyses in spectral regions where analyte and surfactant resonances overlap is avoided. The approach is demonstrated using mixtures of peptides with near‐identical diffusion coefficients. Copyright © 2008 John Wiley & Sons, Ltd.     

T2 relaxation measurement with solvent suppression and implications to solvent suppression in general

A number of suppression pulse sequences including Excitation Sculpting and WATERGATE were incorporated into the standard Carr‐Purcell‐Meiboom‐Gill (CPMG) program for T₂ measurement and experimentally evaluated. The chosen suppression schemes were of varying complexity encompassing pulse program elements, such as presaturation, gradients, and selective pulses, which are typically utilized for solvent suppression. The quality of the spectral data and the accuracy of T₂ measurements of the investigated suppression schemes were evaluated using three aqueous samples with increasing proton content in the water solvent, i.e. by volume 100% D₂O, 80/20% D₂O/H₂O, and 20/80% D₂O/H₂O. For signals removed from the water signal, the T₂ values were generally very consistent between all pulse sequences tested. T₂ measurements can be unreliable for signals too close to the water signal such that they are significantly suppressed as well. Their intensity may actually grow initially through cross relaxation that transfers magnetization back to the solute signal. In turn, this relaxation phenomenon can be exploited to improve the spectral quality of conventional solvent suppression schemes. In favorable cases, even signals that are completely masked by the water signal can be recovered by adding a carefully chosen number of spin echoes with optimized evolution time to conventional water suppression pulse programs, such as Excitation Sculpting or WATERGATE. Copyright © 2009 John Wiley & Sons, Ltd.     

Ultra‐Low‐Field NMR Relaxation and Diffusion Measurements Using an Optical Magnetometer

Nuclear magnetic resonance (NMR) relaxometry and diffusometry are important tools for the characterization of heterogeneous materials and porous media, with applications including medical imaging, food characterization and oil‐well logging. These methods can be extremely effective in applications where high‐resolution NMR is either unnecessary, impractical, or both, as is the case in the emerging field of portable chemical characterization. Here, we present a proof‐of‐concept experiment demonstrating the use of high‐sensitivity optical magnetometers as detectors for ultra‐low‐field NMR relaxation and diffusion measurements.     

NMR Spectroscopic Study of the Self‐Aggregation of 3‐Hexen‐1,5‐diyne Derivatives

The self‐assembly of polycatenar molecules derived from 1,6‐diphenyl‐3,4‐dipropyl‐3‐hexen‐1,5‐diyne has been studied in detail by solution NMR spectroscopy. The analysis of the concentration‐ and temperature‐dependent evolution of the chemical shifts and the diffusion coefficients in [D₁₂]cyclohexane agrees well with an isodesmic model of association in this solvent. The association constants for the stacking and entropy and enthalpy of the process have been obtained. The driving force for the aggregation process is provided by a negative enthalpy (ΔH), which is partially compensated by a negative entropy (ΔS). A structural study of the self‐assembly in solution has been carried out with the help of NOESY NMR spectroscopic experiments.     

Using Diffusion NMR To Characterize Guanosine Self‐Association: Insights into Structure and Mechanism

This paper presents results from a series of pulsed field gradient (PFG) NMR studies on lipophilic guanosine nucleosides that undergo cation‐templated assembly in organic solvents. The use of PFG‐NMR to measure diffusion coefficients for the different aggregates allowed us to observe the influences of cation, solvent and anion on the self‐assembly process. Three case studies are presented. In the first study, diffusion NMR confirmed formation of a hexadecameric G‐quadruplex [G 1 ]₁₆ ? 4 K⁺ ? 4 pic^? in CD₃CN. Furthermore, hexadecamer formation from 5′‐TBDMS‐2′,3′‐isopropylidene G 1 and K⁺ picrate was shown to be a cooperative process in CD₃CN. In the second study, diffusion NMR studies on 5′‐(3,5‐bis(methoxy)benzoyl)‐2′,3′‐isopropylidene G 4 showed that hierarchical self‐association of G₈‐octamers is controlled by the K⁺ cation. Evidence for formation of both discrete G₈‐octamers and G₁₆‐hexadecamers in CD₂Cl₂ was obtained. The position of this octamer–hexadecamer equilibrium was shown to depend on the K⁺ concentration. In the third case, diffusion NMR was used to determine the size of a guanosine self‐assembly where NMR signal integration was ambiguous. Thus, both diffusion NMR and ESI‐MS show that 5′‐O‐acetyl‐2′,3′‐O‐isopropylidene G 7 and Na⁺ picrate form a doubly charged octamer [G 7 ]₈ ? 2 Na⁺ ? 2 pic^? 9 in CD₂Cl₂. The anion's role in stabilizing this particular complex is discussed. In all three cases the information gained from the diffusion NMR technique enabled us to better understand the self‐assembly processes, especially regarding the roles of cation, anion and solvent.     

Sorption and diffusion of methanol and water in PVDF‐g‐PSSA and Nafion® 117 polymer electrolyte membranes

Sorption and diffusion properties of poly(vinylidene fluoride)‐graft‐poly(styrene sulfonic acid) (PVDF‐g‐PSSA) and Nafion® 117 polymer electrolyte membranes were studied in water/methanol mixtures. The two types of membranes were found to have different sorption properties. The Nafion 117 membrane was found to have a maximum in‐solvent uptake around 0.4 to 0.6 mole fraction of methanol, while the PVDF‐g‐PSSA membranes took up less solvent with increasing methanol concentration. The proton NMR spectra were recorded for membranes immersed in deuterated water/methanol mixtures. The spectra showed that the hydroxyl protons inside the membrane exhibit resonance lines different from the resonance lines of hydroxyl protons in the external solvent. The spectral features of the lines of these internal hydroxyl groups in the membranes were different in the Nafion membrane compared with the PVDF‐g‐PSSA membranes. Diffusion measurements with the pulsed field gradient NMR (PFG‐NMR) method showed that the diffusion coefficient of the internal hydroxyl groups in the solvent immersed Nafion membrane mirrors the changes in the diffusion coefficients of hydroxyl and methyl protons in the external solvent. For the PVDF‐g‐PSSA membranes, a decrease in the diffusion coefficient of the internal hydroxyl protons was seen with increasing methanol concentration. These results indicate that the morphology and chemical structure of the membranes have an effect on their solvent sorption and diffusion characteristics. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 3277–3284, 2000     

Unraveling Complex Small‐Molecule Binding Mechanisms by Using Simple NMR Spectroscopy

Heteronuclear NMR spectroscopy provides a unique way to obtain site‐specific information about protein–ligand interactions. Usually, such studies rely on the availability of isotopically labeled proteins, thereby allowing both editing of the spectra and ligand signals to be filtered out. Herein, we report that the use of the methyl SOFAST correlation experiment enables the determination of site‐specific equilibrium binding constants by using unlabeled proteins. By using the binding of L ‐ and D ‐tryptophan to serum albumin as a test case, we determined very accurate dissociation constants for both the high‐ and low‐affinity sites present at the protein surface. The values of site‐specific dissociation constants were closer to those obtained by isothermal titration calorimetry than those obtained from ligand‐observed methods, such as saturation transfer difference. The possibility of measuring ligand binding to serum albumin at physiological concentrations with unlabeled proteins may open up new perspectives in the field of drug discovery.     

Size‐exclusion chromatographic NMR of polymer mixtures

The use of chromatographic stationary phases or solvent modifiers to modulate diffusion properties in NMR experiments is now well established. Their use can be to improve resolution in the diffusion domain or to provide an insight into analyte–modifier interactions and, hence, the chromatography process. Here, we extend previous work using size‐exclusion chromatographic stationary phases to the investigation of polymer mixtures. We demonstrate that similar diffusion modulation behaviour is observed with a size‐exclusion chromatographic stationary phase that can be understood in terms of size‐exclusion behaviour. Copyright © 2014 John Wiley & Sons, Ltd.     

Self‐diffusion coefficients obtained from proton‐decoupled carbon‐13 spectra for analyzing a mixture of terpenes

In the limit of sufficient sensitivity, natural abundance ¹³C offers a much better spectral resolution than proton NMR. This is due to an important chemical shift range and to proton‐decoupling conditions that yield one peak per carbon with practically no overlap. However, pulsed gradient spin echo experiments, which lead to the diffusion coefficient associated with each peak, have scarcely been employed. In this article, we present and compare different ways to access this quantity and we have effectively verified that, without any precaution, diffusion coefficients cannot be properly determined from standard procedures. The cause of such a failure is decoupling during the gradient pulses. We have used a very simple remedy that proved to be very successful on a model mixture of three monoterpenes and that appears as being of general applicability. Copyright © 2009 John Wiley & Sons, Ltd.     

Study of liquid–liquid interfaces by an easily implemented localized NMR sequence

To selectively extract heavy metals from solutions containing fission products, it is essential to optimize the liquid–liquid extraction processes. Such an objective requires improving the fundamental knowledge of the different mechanisms that are involved in these processes. In that respect, we propose a localized NMR sequence named LOCSY to assess the concentration profiles of different species involved in these processes. One of the goals of this sequence is to study the products as close as possible to the liquid–liquid interface with the help of a standard NMR spectrometer of chemistry labs. The one‐dimensional spatial localization along the NMR tube is obtained by a discrete stepping of the frequency‐selective excitation pulses under a pulsed field gradient. Specific data processing has been developed to obtain the 1D NMR spectra as a function of the vertical position in the NMR tube. The LOCSY sequence has been tested and evaluated on three different systems: (i) a cylindrical phantom inserted in the NMR tube containing 4‐methylsalicylic acid solution, (ii) D₂O/olive oil biphasic system, and (iii) the dissolution of solid saccharose in D₂O. These examples illustrate potential applications of the LOCSY sequence, particularly the possibility to measure concentration profiles and to study phenomena such as diffusion, provided the dynamic range is compatible with NMR timescale and sensitivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Gradient‐based solvent suppression methods on a benchtop spectrometer

Benchtop NMR emerges as an appealing alternative to widely extend the scope of NMR spectroscopy in harsh environments and for on‐line monitoring. Obviously, the use of low‐field magnets induces a dramatic reduction of the spectral resolution leading to frequent peak overlaps. This issue is even more serious because applications such as chemical process monitoring involve the use of non‐deuterated solvents, leading to intense and broad peaks overlapping with the signals of interest. In this article, we highlight the need for efficient suppression methods compatible with flowing samples, which is not the case of the common pre‐saturation approaches. Thanks to a gradient coil included in our benchtop spectrometer, we were able to implement modern and efficient solvent suppression blocks such as WET or excitation sculpting to deliver quantitative spectra in the conditions of the on‐line monitoring. While these methods are commonly used at high field, this is the first time that they are investigated on a benchtop setting. Their analytical performance is evaluated and compared under static and on‐flow conditions. The results demonstrate the superiority of gradient‐based methods, thus highlighting the relevance of implementing this device on benchtop spectrometers. The comparison of major solvent suppression methods reveals an optimum performance for the WET‐180‐NOESY experiment, both under static and on‐flow conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

Self‐diffusion in a hyaluronic acid–albumin–water system as studied by NMR

Hyaluronic acid (HA) is an anionic biopolymer that is present in many tissues and can be involved in cancerous neoformations. HA can form complexes with proteins (particularly, serum albumin) in the body. However, HA structures and processes involving HA have not been extensively studied by NMR because the molecule's rigid structure makes these studies problematic. In the current work, self‐diffusion of HA and bovine serum albumin (BSA), and water in solutions was measured by ¹H pulsed field gradient NMR (PFG NMR) with a focus on the HA‐BSA‐D₂O systems at various concentrations of BSA and HA. It was shown that in the presence of even a small amount of HA, the self‐diffusion coefficient (SDC) of BSA decreases. To explain this fact, three hypotheses were proposed and analyzed. The first one was based on the effect of slowing down of water mobility in the presence of HA. The second hypothesis suggested an effect of mechanical collisions of BSA with HA molecules. The third hypothesized that BSA and HA molecules form a complex where BSA molecules reduced in mobility. It was shown that the third mechanism is the most likely. The state of the BSA molecules in the BSA‐HA‐D₂O system corresponds to a ‘fast exchange’ condition from the NMR point of view: BSA molecules reside in the ‘free’ and ‘bound’ (with HA) states for much shorter time than the diffusion time of the PFG NMR experiment, 7 ms. The fractions of ‘bound’ BSA molecules in the BSA‐HA complex were estimated. Copyright © 2012 John Wiley & Sons, Ltd.     

Prediction of solvent diffusion coefficient in amorphous polymers based on an equation‐of‐state

This study develops a modified free‐volume model to predict solvent diffusion coefficients in amorphous polymers by combining the Vrentas–Duda model with the Simha–Somcynsky (S‐S) equation‐of‐state (EOS), and all the original parameters can be used in the modified model. The free volume of the polymer is estimated from the S‐S EOS together with the Williams‐Landel‐Ferry fractional free volume, and the complex process of determining polymer free‐volume parameters in the Vrentas–Duda model and measuring polymer viscoelasticity can be avoided. Moreover, the modified model includes the influence of not only temperature but also pressure on solvent diffusivity. Three common polymers and four solvents are employed to demonstrate the predictions of the modified model. The calculation results are generally consistent with the experimental values. It is reasonable to expect that the modified free‐volume model will become a useful tool in polymer process development. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 1000–1009, 2006     

Offset‐compensated and zero‐quantum suppressed ROESY provides accurate 1H–1H distances in small to medium‐sized molecules

A robust version of the off‐resonance ROESY pulse scheme is suggested for the measurement of proton–proton distances or slow chemical exchange in small to medium‐sized molecules. The method implements adiabatic ramps to establish a pair of opposite frequency off‐resonance spin lock fields – with optionally randomized duration – and adiabatic inversion pulses with simultaneous gradients for efficient zero‐quantum suppression. The amended pulse sequence yields pure absorption cross‐peaks and works safely for small to medium‐sized molecules. The applicability of the method has been demonstrated using small, rigid molecules (strychnine and codeine) and was also applied for a cyclic peptide and a small protein. We found that the pure phase cross‐peaks of the new ROESY version are beneficial for distance measurements. The one‐dimensional (selective) version of the new method is also powerful for measuring selected pair‐wise interactions and distance determination. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular dynamics and composition of crude oil by low‐field nuclear magnetic resonance

Nuclear magnetic resonance (NMR) techniques are widely used to identify pure substances and probe protein dynamics. Oil is a complex mixture composed of hydrocarbons, which have a wide range of molecular size distribution. Previous work show that empirical correlations of relaxation times and diffusion coefficients were found for simple alkane mixtures, and also the shape of the relaxation and diffusion distribution functions are related to the composition of the fluids. The 2D NMR is a promising qualitative evaluation method for oil composition. But uncertainty in the interpretation of crude oil indicated further study was required. In this research, the effect of each composition on relaxation distribution functions is analyzed in detail. We also suggest a new method for prediction of the rotational correlation time distribution of crude oil molecules using low field NMR (LF‐NMR) relaxation time distributions. A set of down‐hole NMR fluid analysis system is independently designed and developed for fluid measurement. We illustrate this with relaxation–relaxation correlation experiments and rotational correlation time distributions on a series of hydrocarbon mixtures that employ our laboratory‐designed downhole NMR fluid analyzer. The LF‐NMR is a useful tool for detecting oil composition and monitoring oil property changes. Copyright © 2016 John Wiley & Sons, Ltd.