Simultaneous measurements of T1 and T2 during fast polymerization reaction using continuous wave‐free precession NMR method

Continuous wave‐free precession (CWFP) pulse sequence employing time domain nuclear magnetic resonance spectroscopy (TD‐NMR) was used to measure longitudinal (T₁) and transverse relaxation times (T₂), during the cure of a commercial epoxy resin (Araldite^TM) with a 10‐min solidification time. The intensity of the NMR signal after the first pulse and in the CWFP regime were used to monitor the concentration of the monomers, and the relaxation times were used to monitor the chain mobility. The main advantage of CWFP over the standard methods to measure relaxation times, inversion recovery (inv‐rec) for T₁ and Carr‐Purcell‐Meiboom‐Gill (CPMG) for T₂, is that the measurement of both relaxation times can be performed in a fast and single NMR experiment and, therefore, using a single reaction batch. CWFP is also as fast as the CPMG measurement but at least fivefold faster than the method to obtain T₁ using null point approximation in the inv‐rec method. Therefore, the CWFP sequence can be used as a fast and general method to measure relaxation times in polymerization reactions, even with fast solidification time. As a TD‐NMR technique, CWFP can be employed in any low‐cost bench top TD‐NMR equipment commonly used in an academic or industrial laboratory. Copyright © 2012 John Wiley & Sons, Ltd.     

Processing of high resolution magic angle spinning spectra of breast cancer cells by the filter diagonalization method

Proton nuclear magnetic resonance ((1)H NMR) spectroscopy for detection of biochemical changes in biological samples is a successful technique. However, the achieved NMR resolution is not sufficiently high when the analysis is performed with intact cells. To improve spectral resolution, high resolution magic angle spinning (HR-MAS) is used and the broad signals are separated by a T(2) filter based on the CPMG pulse sequence. Additionally, HR-MAS experiments with a T(2) filter are preceded by a water suppression procedure. The goal of this work is to demonstrate that the experimental procedures of water suppression and T(2) or diffusing filters are unnecessary steps when the filter diagonalization method (FDM) is used to process the time domain HR-MAS signals. Manipulation of the FDM results, represented as a tabular list of peak positions, widths, amplitudes and phases, allows the removal of water signals without the disturbing overlapping or nearby signals. Additionally, the FDM can also be used for phase correction and noise suppression, and to discriminate between sharp and broad lines. Results demonstrate the applicability of the FDM post-acquisition processing to obtain high quality HR-MAS spectra of heterogeneous biological materials.     

Improvements in lipid suppression for 1H NMR-based metabolomics: Applications to solution-state and HR-MAS NMR in natural and in vivo samples

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. In solution-state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr-Purcell-Meiboom-Gill sequence (CPMG) train is replaced with a low-power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR-MAS as well as static solution-state samples. Applications are also extended to include in vivo organisms. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high-lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.     

Sensitivity enhancement for maximally resolved two‐dimensional NMR by nonuniform sampling

Resolving NMR signals which are separated in frequency on the order of their line widths requires obtaining the time domain free induction decay for a maximum time t_max = πT₂, where T₂ is the transverse relaxation time of the given signals. Unfortunately, samples acquired beyond ～1.26T₂ contribute more noise than signal to the data; and samples in the range of about (0.75–1.26)× T₂ have a negligible effect on the signal‐to‐noise ratio (SNR). Therefore, one must sacrifice SNR to reach evolution times of πT₂. One can preserve resolution in a shorter total experimental time by selecting a reduced set of samples from the Nyquist grid according to an exponential probability density which is on the order of the T₂ of the signals. This practice is widely termed nonuniform sampling (NUS). We derive analytic theory for the enhancement of the intrinsic SNR of NUS time domain data compared with uniformly sampled data when the total experimental times are equivalent. This theory is general for any t_max and exponential weighting and is further carefully validated with simulations. Enhancements of SNR in the time domain on the order of twofold are routinely available when t_max ～ πT₂ and are reflected in the subsequent maximum entropy reconstructed spectra. SNR enhancement by NUS is demonstrated to be helpful in enabling the acquisition of HMQC spectra of dilute bile salts in which high resolution in the indirect carbon dimension is required. Copyright © 2011 John Wiley & Sons, Ltd.     

Qualitative analysis by online nuclear magnetic resonance using Carr-Purcell-Meiboom-Gill sequence with low refocusing flip angles

The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence has been used in many applications of magnetic resonance imaging (MRI) and low-resolution NMR (LRNMR) spectroscopy. Recently, CPMG was used in online LRNMR measurements that use long RF pulse trains, causing an increase in probe temperature and, therefore, tuning and matching maladjustments. To minimize this problem, the use of a low-power CPMG sequence based on low refocusing pulse flip angles (LRFA) was studied experimentally and theoretically. This approach has been used in several MRI protocols to reduce incident RF power and meet the specific absorption rate. The results for CPMG with LRFA of 3π/4 (CPMG₁₃₅), π/2 (CPMG₉₀) and π/4 (CPMG₄₅) were compared with conventional CPMG with refocusing π pulses. For a homogeneous field, with linewidth equal to Δυ = 15 Hz, the refocusing flip angles can be as low as π/4 to obtain the transverse relaxation time (T₂) value with errors below 5%. For a less homogeneous magnetic field, Δυ = 100 Hz, the choice of the LRFA has to take into account the reduction in the intensity of the CPMG signal and the increase in the time constant of the CPMG decay that also becomes dependent on longitudinal relaxation time (T₁). We have compared the T₂ values measured by conventional CPMG and CPMG₉₀ for 30 oilseed species, and a good correlation coefficient, r = 0.98, was obtained. Therefore, for oilseeds, the T₂ measurements performed with π/2 refocusing pulses (CPMG₉₀), with the same pulse width of conventional CPMG, use only 25% of the RF power. This reduces the heating problem in the probe and reduces the power deposition in the samples.     

Paramagnetic NMR relaxation in polymeric matrixes: sensitivity enhancement and selective suppression of embedded species (1H and 13C PSR filter)

A study of the practical applications of the addition of paramagnetic spin relaxation (PSR) ions to a variety of polymers (PLL, PAA, PGA, PVP, and polysaccharides such as hyaluronic acid, chitosan, mannan, and dextran) in solution (D2O and DMSO-d6) is described. Use of Gd(III), Cu(II), and Mn(II) allows a reduction of up to 500% in the 1H longitudinal relaxation times (T1), and so in the time necessary for recording quantitative NMR spectra (sensitivity enhancement) neither an increase of the spectral line width nor chemical shift changes resulted from addition of any of the PSR agents tested. Selective suppression of the 1H and 13C NMR signals of certain components (low MW molecules and polymers) in the spectrum of a mixture was attained thanks to their different sensitivity [transverse relaxation times (T2)] to Gd(III) (PSR filter). Illustration of this strategy with block copolymers (PGA-g-PEG) and mixtures of polymers and low MW molecules (i.e., lactose-hyaluronic acid, dextran-PAA, PVP-glutamic acid) in 1D and 2D NMR experiments (COSY and HMQC) is presented. In those mixtures where PSR and CPMG filters alone failed in the suppression of certain components (i.e., PVP-mannan-hyaluronic acid) due to their similarity of 1H T2 values and sensitivities to Gd(III), use of the PSR filter in combination with CPMG sequences (PSR-CPMG filter) successfully resulted in the sequential suppression of the components (hyaluronic acid first and then mannan).     

Rapid parameter optimization of low signal‐to‐noise samples in NMR spectroscopy using rapid CPMG pulsing during acquisition: application to recycle delays

A method is presented that combines Carr–Purcell–Meiboom–Gill (CPMG) during acquisition with either selective or nonselective excitation to produce a considerable intensity enhancement and a simultaneous loss in chemical shift information. A range of parameters can theoretically be optimized very rapidly on the basis of the signal from the entire sample (hard excitation) or spectral subregion (soft excitation) and should prove useful for biological, environmental, and polymer samples that often exhibit highly dispersed and broad spectral profiles. To demonstrate the concept, we focus on the application of our method to T₁ determination, specifically for the slowest relaxing components in a sample, which ultimately determines the optimal recycle delay in quantitative NMR. The traditional inversion recovery (IR) pulse program is combined with a CPMG sequence during acquisition. The slowest relaxing components are selected with a shaped pulse, and then, low‐power CPMG echoes are applied during acquisition with intervals shorter than chemical shift evolution (RCPMG) thus producing a single peak with an SNR commensurate with the sum of the signal integrals in the selected region. A traditional ¹³C IR experiment is compared with the selective ¹³C IR‐RCPMG sequence and yields the same T₁ values for samples of lysozyme and riverine dissolved organic matter within error. For lysozyme, the RCPMG approach is ~70 times faster, and in the case of dissolved organic matter is over 600 times faster. This approach can be adapted for the optimization of a host of parameters where chemical shift information is not necessary, such as cross‐polarization/mixing times and pulse lengths. Copyright © 2013 John Wiley & Sons, Ltd.     

A NMR reverse diffusion filter for the simplification of spectra of complex mixtures and the study of drug receptor interactions

A reverse diffusion filter NMR experiment (Drev) is proposed for the study of small molecules in binding with macromolecules. The filtering efficiency of Drev to eliminate the signals of the macromolecule is shown to be superior to conventional transverse relaxation filters at least for macromolecules containing a significant fraction of flexible residues. The Drev filter was also a useful complement for ligand‐based NMR screening in combination with saturation transfer difference experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

The direct evaluation of dissociation constants (K_D) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor–ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K_D, as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor–ligand dissociation constants (K_D) from single‐ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein–ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein–ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein–ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well‐studied protein–ligand systems: the binding of the saccharides GlcNAc and GlcNAcβ1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L ‐tryptophan to bovine serum albumin (BSA). In all cases, the experimental K_D measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein–ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.     

Water and oil signal assignment in low-moisture mozzarella as determined by time-domain NMR T2 relaxometry

A time-domain ¹H nuclear magnetic resonance relaxometry method was elaborated for the rapid microstructural characterization of mozzarella cheese. For this purpose, there is a strong need to know how the experimentally determined T₂ relaxation time distribution can be related to specific constituents in mozzarella. In this study, a detailed investigation is offered for fresh and aged low-moisture mozzarella cheese, often applied as a pizza cheese, by application of both a conventional Carr–Purcell–Meiboom–Gill (CPMG) sequence and a free-induction decay CPMG (FID-CPMG) sequence. The relaxation behavior was further elucidated by addition of deuterium oxide and by mild heat treatment of samples. The relaxation times of water protons in mozzarella were found to range from a few microseconds to some tens of milliseconds (in aged mozzarella) or to about hundred milliseconds (in fresh mozzarella). The upper limit of the T₂ distribution can even be extended to the seconds range upon releasing water protons from the mozzarella matrix using a mild heat treatment or upon addition of deuterated water. Both stimuli also provided evidence for the absorption of water into the cheese matrix. The potential release and uptake of water demonstrated that mozzarella acts as a very dynamic system during production and storage. The detected differences in the behavior of the water fraction between fresh and aged low-moisture mozzarella might be utilized to study the influence of either production and/or storage conditions on the cheese ripening process.     

Extending the Sensitivity of CEST NMR Spectroscopy to Micro-to-Millisecond Dynamics in Nucleic Acids Using High-Power Radio-Frequency Fields

Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr–Purcell–Meiboom–Gill (CPMG), chemical exchange saturation transfer (CEST), and R_1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R_1ρ experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high-power radio-frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high-power fields in CEST, this should make it easier to characterize micro-to-millisecond dynamics in biomolecules.     

UV–vis,IR and 1H NMR spectroscopic studies and characterization of ionic-pair crystal violet–oxytetracycline

The present study shows the formation and characterization of the ionic-pair between the antibiotic oxytetracycline and the dye crystal violet in ammonia solution pH 9.0 ± 0.2 extracted into chloroform. The characterization was demonstrated using UV–vis spectrophotometry, ¹H NMR, measurement of relaxation times T₁ and IR spectroscopy, using a comparison between the signals of individual pure compounds with the signals with the mixture CV–OTC in different alkaline media. The formation of ionic-pair was also corroborated by new signals and chemical shifts. (2D) NMR spectroscopy experiments show that the interaction is electrostatic.     

Site Covalent Modification of Methionyl Peptides for the Production of FRET Complexes

Flax cyclic peptides (orbitides, linusorbs (LOs)) [1–8‐NαC],[1‐MetO₂]‐linusorb B1 ([MetO₂]‐LO1) and [1–9‐NαC],[1‐MetO₂]‐linusorb B2 ([MetO₂]‐LO2) are biologically active. These LOs lack active nuclei commonly used in peptide modification. We have developed reactions to activate methionine methyl sulphide to produce stable derivatives. In these reactions, LOs are converted to sulfonium intermediates and subsequently to derivatives containing active nuclei while preserving their fundamental structures. The reaction conditions preserved cyclic peptide fundamental structure and organic solvent solubility. [Met]‐LO1 and [Met]‐LO2 analogues containing activated groups (?CN, ?COOEt, and ?NH₂) in the form of methionine, methionine (S)‐oxide, and methionine (S,S)‐dioxide amino acids were synthesized and characterized by LCMS and 1D and 2D NMR spectroscopy. Coumarin orbitide complexes produced in this manner bind Eu³⁺ yielding FRET compounds that absorb energy through coumarin antennae and emit photons at lanthanide wavelengths.     

Proton magnetic relaxation study of pretransitional phenomena in the isotropic phase of a nematic liquid crystal I. Dynamics of local order fluctuations

Nuclear magnetic relaxation was investigated in a broad temperature region above the clearing point (T_c) of a nematic liquid crystal. Dependence of spin-spin relaxation time on the pulse interval observed in the Curr-Purcell-Meiboom-Gill (CPMG) experiment indicates an exchange of nuclei between the states differing in local magnetic fields. By fitting of the Luz-Meiboom equation to the CPMG results, the mean lifetime of sites and modulation frequency δω were determined. The rather slow exchange (lifetimes changing with temperature in the range 20-90 ms) is suggested as manifesting the local order fluctuations in the pretransitional zone of the LC. A simple two-site model of a pretransitional zone was considered (cluster ? isotropic surrounding). Dipole-dipole interactions in clusters are unaveraged due to the local ordering, whereas in the isotropic subphase local magnetic fields are motionally averaged. Therefore, local order fluctuations are accompanied by the exchange observed in the CPMG sequence. Correspondence of the temperature dependence of δω to the Curie-Weiss law was established: δω^-2 ∝ T - T*, (T* = T_c - 1), thus providing proof of our interpretation.     

Nuclear magnetic relaxation in polyolefin resins

Nuclear magnetic resonance (NMR) spin–lattice relaxation times (T₁) in various polyethylene and polypropylene resins were measured at 20 MHz and at temperatures of 130–490 K. At each temperature and for all resins, only a single value of T₁ was found, which was consistent with the occurrence of rapid spin diffusion throughout the protons attached to the polymer chains. The data were analyzed for the estimation of activation energies corresponding to molecular motion causing spin–lattice relaxation. Two well‐defined minima were found for log_e(T₁) plotted as a function of temperature for all of the polypropylene resins. Single very broad minima were found for all of the polyethylene samples. In contrast, the free induction decay signals from all of the resins following single radio‐frequency pulses were observed to contain a rapidly decaying component followed by a much more slowly decaying signal. These components were used to estimate the amount of rigid component present in the solid resins at room temperature. Samples of one high‐density polyethylene and one low‐density polyethylene were irradiated with γ radiation up to a 500‐kGy dose to examine the effects of crosslinking on the NMR relaxation. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 572–584, 2002; DOI 10.1002/polb.10116     

Behavior of Two Almost Identical Spins during the CPMG Pulse Sequence

Multiple‐spin‐echo experiments have found wide use in nuclear magnetic resonance spectroscopy. In particular, the Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence is used to determine transverse relaxation times T₂. Herein it is demonstrated, both theoretically and experimentally, that for a pair of almost identical spins‐1/2 the experimental setup can have a profound effect on the observed spin dynamics. It is shown that, in the case of dipolar relaxation, the measured T₂ values can roughly vary between the limits of identical and unlike spins, just depending on the repetition rate of π pulses with respect to chemical shift separation. Such an effect can, in the extreme narrowing regime, amount to a 50 % difference.     

Studies on the phase structure of ethylene‐vinyl acetate copolymers by solid‐state 1H and 13C NMR

The phase structure of a series of ethylene‐vinyl acetate copolymers has been investigated by solid‐state wide‐line ¹H NMR and solid‐state high‐resolution ¹³C NMR spectroscopy. Not only the degree of crystallinity but the relative contents of the monoclinic and orthorhombic crystals within the crystalline region varied with the vinyl acetate (VA) content. Biexponential ¹³C NMR spin–lattice relaxation behavior was observed for the crystalline region of all samples. The component with longer ¹³C NMR spin–lattice relaxation time (T₁) was attributed to the internal part of the crystalline region, whereas the component with shorter ¹³C NMR T₁ to the mobile crystalline component was located between the noncrystalline region and the internal part of the crystalline region. The content of the mobile crystalline component relative to the internal part of the crystalline region increased with the VA content, showing that the ¹³C NMR spin–lattice relaxation behavior is closely related to the crystalline structure of the copolymers. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 2199–2207, 2002     

Extending the Sensitivity of CEST NMR Spectroscopy to Micro‐to‐Millisecond Dynamics in Nucleic Acids Using High‐Power Radio‐Frequency Fields

Biomolecules undergo motions on the micro‐to‐millisecond timescale to adopt low‐populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr–Purcell–Meiboom–Gill (CPMG), chemical exchange saturation transfer (CEST), and R_1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R_1ρ experiment are more broadly sensitive to motions on the micro‐to‐millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high‐power radio‐frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro‐to‐millisecond timescale. Given the ease of implementing high‐power fields in CEST, this should make it easier to characterize micro‐to‐millisecond dynamics in biomolecules.