High-resolution NMR Spectra Research Articles

Intermolecular single-quantum coherence sequences for high-resolution NMR spectra in inhomogeneous fields

A new pulse sequence based on intermolecular single-quantum coherences (iSQCs) is proposed to obtain high-resolution NMR spectroscopy in inhomogeneous magnetic fields via fast 2D acquisition. Taking the intrinsic properties of iSQCs, the sequence is time-efficient with a narrow spectral width in the indirect dimension. It can recover useful information of chemical shifts, relative peak areas, J coupling constants, and multiplet patterns even when the field inhomogeneity is severe enough to erase almost all spectroscopic information. Moreover, good solvent suppression efficiency can be achieved by this sequence even with imperfect radio-frequency pulse flip angles. Spatially localized iSQC spectroscopy was performed on a sample packed with pig brain tissue and cucumber to show the feasibility of the sequence in in vivo magnetic resonance spectroscopy (MRS). This sequence may provide a promising way for the applications on in vivo and in situ high-resolution NMR spectroscopy.

The influence of sulfur on the structural-dynamic parameters of petroleum systems studied by the NMR technique

A number of crude oils, fuel oils, and asphalts containing sulfur with a concentration in the range of 0.16–4.61 wt % were studied by the nuclear magnetic resonance technique. The NMR relaxation parameters were measured with a 08/PC laboratory NMR relaxometer made in-house according to TU 25-4823764.0031-90 and a computerized portable proton magnetic resonance analyzer. A multiexponential character of the spinecho envelope was revealed for all samples, which was deconvoluted into three exponentials having relaxation times of T 2A, T 2B, and T 2C. The obtained high-resolution NMR spectra of crude oil, fuel oil, and asphalt show that sulfur contributes to the width of all spectral lines. The slope of the T 1A, T 1B, T 1C, T 2A, T 2B, and T 2C plots varies depending on the temperature range of the measurement in all cases, thereby indicating a change in the activation energy E Ai of molecular motion. All of the activation energies increase with an increase in the sulfur concentration. Over the entire range of measurement temperatures, there is one activation energy for crude oils and two activation energies E A 1 and E A 2 for fuel oils, with the low-temperature activation energies having higher values. An analysis of the asphalt revealed five, not two, activation energies or intervals of constant E B 2 values (structurally-dynamically ordered phases of the shells of asphalt structure units).

Network structures and dynamics of dry and swollen poly(acrylate)s. Characterization of high- and low-frequency motions as revealed by suppressed or recovered intensities (SRI) analysis of 13C NMR

We recorded temperature-dependent high-resolution 13C NMR spectra of dry and swollen poly(acrylate)s [poly(2-methoxyethyl acrylate) (PMEA), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(tetrahydrofurfuryl acrylate) (PTHFA)] by dipolar decoupled-magic angle spinning (DD-MAS) and cross-polarization-magic angle spinning (CP-MAS) methods, to gain insight into their network structures and dynamics. Suppressed or recovered intensities (SRI) analysis of 13C CP-MAS and DD-MAS NMR was successfully utilized, to reveal portions of dry and swollen polymers which undergo fast and slow motions with fluctuation frequencies in the order of 10 8 Hz and 10 4–10 5 Hz, respectively. Fast isotropic motions with frequency higher than 10 8 Hz at ambient temperature were located to the portions in which 13C CP-MAS NMR signals of swollen PMEA were selectively suppressed. In contrast, low-frequency motion was identified to the portions in which 13C DD-MAS (and CP-MAS) signals are most suppressed at the characteristic suppression temperature(s) T s. Network of PMEA gels (containing 7 wt% of water) turns out to be formed by partial association of backbones only, as manifested from their T s gradient at lowered temperature, whereas networks of PHEMA (containing 40 wt% of water) and PTHFA (9 wt% of water) gels are tightly formed through mutual inter-chain associations of both backbones and side-chains, as viewed from the raised T s values for both near at ambient temperature. It is also interesting to note that flexibility of gel network (PMEA > PTHFA > PHEMA) characterized by the suppression temperature T s (PMEA < PTHFA < PHEMA) is well related with a characteristic parameter for biocompatibility such as the production of TAT (thrombin–antithrombin III complex) as a marker of activation of the coagulation system.

Intermolecular double-quantum coherence NMR spectroscopy in moderate inhomogeneous fields

Intermolecular multiple-quantum coherences (iMQCs) can be utilized to retrieve high-resolution NMR spectra in inhomogeneous magnetic fields. The application of selective pulses in pulse sequences can greatly simplify 2D iMQC spectra. However, so far high-resolution iMQC methods are mainly used in relatively small field inhomogeneities. In this paper, we took the IDEAL-II sequence as an example to study their applicability in moderate inhomogeneous magnetic fields. The experimental and simulation results show that high-resolution NMR spectra can be obtained in moderate inhomogeneous fields if the excitation range of selective pulse is properly set. Once the field inhomogeneity reaches a certain degree, the appearance of undesirable intermolecular cross-peaks due to the distant dipolar field produced by solute spins is inevitable. The spectral quality may vary with sample even in the same moderate inhomogeneous fields, depending on the chemical shift distributions and the J coupling networks of the components under study. The conclusions drawn in this paper are generally applicable to all high-resolution iMQC methods utilizing selective RF pulses.

Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane beta-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.

Synthesis and structural characterization of pyrazol-1′-ylpyrazolo[1,5- a]pyrimidines by multinuclear NMR spectroscopy

3(5)-Amino-5(3)-hydrazinopyrazole dihydrochloride ( 8) reacts with pentane-2,4-dione ( 9a) to afford 2-(3′,5′-dimethylpyrazol-1′-yl)-5,7-dimethylpyrazolo[1,5- a]pyrimidine ( 10a) instead of the 3-(3′,5′-dimethylpyrazol-1′-yl)-4,6-dimethyl-1 H-pyrazolo[3,4- b]pyridine isomer ( 11). Similarly, the reaction of 8 with phenyl-1,3-butanedione ( 9b) resulted into the formation of 2-(3′-methyl-5′-phenylpyrazol-1′-yl)-5-methyl-7-phenylpyrazolo[1,5- a]pyrimidine ( 10b) out of the four possible regioisomers. The structure of the reaction products 10a and b was established by analysis of high-resolution 1H NMR spectra. Complete spectral analysis has been achieved utilizing ( 1H– 13C) HMQC as well as ( 1H– 13C) and ( 1H– 15N) HMBC experiments.

Preferential Clustering of Water Molecules During Hydration of the Ammonium Form of the Solid Acid Catalyst ZSM-5

Hydration of the ammonium form of the solid acid catalyst ZSM-5 is investigated by applying a technique developed recently for in situ solid-state NMR studies of adsorption processes. From high-resolution solid-state 1H NMR spectra recorded as a function of time during the hydration process, insights are established concerning the nature of the interaction between the adsorbed water molecules and the ammonium cations in the ZSM-5 host material. In particular, quantitative information is established regarding the clustering of water molecules during the hydration process, and indicates the preferential formation of clusters comprising two water molecules associated with each hydrated ammonium cation.

High-resolution solid-state NMR of anisotropically mobile molecules under very low-power 1H decoupling and moderate magic-angle spinning

We show that for observing high-resolution heteronuclear NMR spectra of anisotropically mobile systems with order parameters less than 0.25, moderate magic-angle spinning (MAS) rates of ∼11 kHz combined with 1H decoupling at 1–2 kHz are sufficient. Broadband decoupling at this low 1H nutation frequency is achieved by composite pulse sequences such as WALTZ-16. We demonstrate this moderate MAS low-power decoupling technique on hydrated POPC lipid membranes, and show that 1 kHz 1H decoupling yields spectra with the same resolution and sensitivity as spectra measured under 50 kHz 1H decoupling when the same acquisition times (∼50 ms) are used, but the low-power decoupled spectra give higher resolution and sensitivity when longer acquisition times (>150 ms) are used, which are not possible with high-power decoupling. The limits of validity of this approach are explored for a range of spinning rates and molecular mobilities using more rigid membrane systems such as POPC/cholesterol mixed bilayers. Finally, we show 15N and 13C spectra of a uniaxially diffusing membrane peptide assembly, the influenza A M2 transmembrane domain, under 11 kHz MAS and 2 kHz 1H decoupling. The peptide 15N and 13C intensities at low-power decoupling are 70–80% of the high-power decoupled intensities. Therefore, it is possible to study anisotropically mobile lipids and membrane peptides using liquid-state NMR equipment, relatively large rotors, and moderate MAS frequencies.

Specific Solvation Interactions of CO2 on Acetate and Trifluoroacetate Imidazolium Based Ionic Liquids at High Pressures

New classes of acidic or basic ionic liquids (ILs) are gaining special attention, since the efficiency of many processes can be enhanced by the judicious manipulation of these properties. The absorption of sour gases can be enhanced by the basic character of the IL. The fluorination of the cation or the anion can also contribute to enhance the gas solubility. In this work these two characteristics are evaluated through the study of the gas-liquid equilibrium of two ionic liquids based on similar anions, 1-butyl-3-methylimidazolium acetate ([C4mim][Ac]) and 1-butyl-3-methylimidazolium trifluoroacetate ([C4mim][TFA]), with carbon dioxide (CO2) at temperatures up to 363 K and pressures up to 76 MPa. The data reported are shown to be thermodynamically consistent. Henry's constants estimated from the experimental data show the solubility of CO2 on the [C4mim][Ac] to be spontaneous unlike in [C4mim][TFA] due to the differences in solvation enthalpies in these systems. Ab initio calculations were performed on simple intermolecular complexes of CO2 with acetate and trifluoroacetate using MP2/6-31G(d) and the G3 and G3MP2 theoretical procedures to understand the interactions between CO2 and the anions. The theoretical study indicates that although both anions exhibit a simultaneous interaction of the two oxygen of the carboxylate group with the CO2, the acetate acts as a stronger Lewis base than the trifluoroacetate. 13C high-resolution and magic angle spinning (HRMAS) NMR spectra provide further evidence for the acid/base solvation mechanism and the stability of the acetate ion on these systems. Further similarities and differences observed between the two anions in what concerns the solvation of CO2 are discussed.

Deciphering the NMR Fingerprints of the Disordered System with Quantum Chemical Studies

Recent developments in solid-state NMR techniques helped acquire high-resolution NMR spectra for solid systems with structural disorder. But the structural origin of the observed chemical shift nonequivalence in these systems has not been revealed. We report a quantum chemical investigation of the solid-state NMR spectrum in N,N-bis(diphenylphosphino)-N-((S)-alpha-methylbenzyl)amine, where eight nonequivalent (31)P NMR chemical shifts were resolved with a range of 13.0 ppm. Results from using different quantum chemical methods, computational algorithms, intermolecular effects, and structures indicate that for the disordered system, geometry optimization gives the best accord with experimental NMR chemical shifts, which has a theory-versus-experiment correlation R(2) = 0.949 and SD = 1.1 ppm, or R(2) = 0.994 and SD = 0.4 ppm when the average of two unassigned NMR shifts for each molecule is used. In addition, these calculations indicate that the experimental chemical shift nonequivalence in this system is mainly a consequence of the different geometries around the phosphorus atoms due to disordered environments. The experimental (31)P NMR chemical shifts are well correlated (R(2) = 0.981) with two conformation angles and one bond length, each associated with one of the three bonding interactions around the phosphorus atoms. These results will facilitate the use of quantum chemical techniques in structural characterization of disordered solids and elucidation of NMR properties.

Protein structure determination in living cells by in-cell NMR spectroscopy

Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

Fast acquisition of high-resolution NMR spectra in inhomogeneous fields via intermolecular double-quantum coherences

A pulse sequence, IDEAL-II, is proposed based on the concept of intermolecular dipolar-interaction enhanced all lines [Z. Chen et al., J. Am. Chem. Soc. 126, 446 (2004)] for obtaining one-dimensional (1D) high-resolution liquid NMR spectra in inhomogeneous fields via two-dimensional acquisitions. With the new acquisition scheme, the range of magnetic field inhomogeneity rather than chemical shift is sampled in the indirect dimension. This enables a great reduction in acquisition time and amount of data, much improved over the original IDEAL implementation. It is applicable to both isolated and J-coupled spin systems in liquid. For the latter, apparent J coupling constants are magnified threefold in spectra obtained with this sequence. This allows a more accurate measurement of J coupling constants in the cases of small J coupling constants or large inhomogeneous fields. Analytical expression was derived based on intermolecular multiple-quantum coherence treatments. Solution samples that were purposely deshimmed and biological samples with intrinsic field inhomogeneities were tested. Experimental results demonstrate that this sequence retains useful structural information including chemical shifts, relative peak areas, and multiplet patterns of J coupling even when the field inhomogeneity is severe enough to almost erase all spectroscopic information with conventional 1D single-quantum coherence techniques. This sequence is more applicable to weakly coupled and uncoupled spin systems, potentially useful for studying metabolites in in vivo NMR spectroscopy and for characterizing technologically important new materials in combinatorial chemistry.

High-resolution NMR spectroscopy in inhomogeneous fields via heteronuclear intermolecular multiple-quantum coherences

A heteronuclear CRAZED pulse sequence was utilized to obtain high-resolution NMR spectra via heteronuclear intermolecular zero-quantum coherences (iZQCs) or intermolecular double-quantum coherences (iDQCs) in inhomogeneous fields. It overcomes the difficulty of selective excitation in homonuclear CRAZED experiments when the chemical shifts of solvent and solute are close to each other. In the 1D spectrum extracted from the 2D data, chemical shifts, coupling constants, multiplet patterns, and relative peak areas are almost independent of the magnetic field inhomogeneity. Theoretical predictions were verified by experimental observations.

High-Resolution Structure of Piscidin in Aligned Lipid Bilayers: Implications for Antimicrobial Mode of Action

Piscidin is an amphipathic cationic antimicrobial peptide that belongs to a large family of versatile host-defense peptides, which interact, at least initially, with cell membranes in order to perform their function. In the research presented here, we characterize the secondary structure and dynamics of piscidin in order to identify factors optimizing specific molecular interactions that are directly related to its function and mode of action. Our long term goal is to identify common principles that will facilitate the design of pharmaceuticals with broad-spectrum antibacterial activity, minimum induction of bacterial resistance, and low toxicity for mammalian cells.Previously, we demonstrated that membrane-bound piscidin 1 (p1) adopts an alpha-helical structure, which lies in the plane of the bilayer and experiences fast motions. Here, high-resolution solid-state NMR spectra have been obtained from multiply 15N-backbone-labeled p1 aligned in hydrated lipid bilayers. Analysis of data from twenty sites of this 22-mer reveals two helical segments separated by a kink at Gly13. This kink may help one portion of the peptide insert more deeply in the hydrophobic lipid bilayer, which may be related to the mechanism of membrane disruption. To characterize water exposure, hydrogen-deuterium exchange experiments have been performed on 15N-backbone labeled samples. In addition, solid-state NMR was applied to 15N-side-chain-labeled His-17 p1 to titrate this side chain, which resides at the interface between the hydrophilic and hydrophobic domains of p1. Overall, our atomic-level investigation of the structure, dynamics, and water exposure of membrane-bound piscidin provides new insights into its mode of action and thus help understand how antimicrobial peptides recognize membranes and initiate their activities on microbial cells.

Combining STMAS and CRAMPS NMR spectroscopy: High-resolution HETCOR NMR spectra of quadrupolar and 1H nuclei in solids

High-resolution two-dimensional (2D) heteronuclear correlation spectra between half-integer quadrupolar and 1H nuclei have been obtained combining Satellite Transition Magic-Angle Spinning (STMAS) and wPMLG3 CRAMPS methods. The experiments use split-t 1 versions of DQ/DQF-STMAS adapted to 3/2 and 5/2 spins to obtain a 23Na and 27Al isotropic dimension, respectively. Transfer of magnetization from quadrupolar nuclei to protons has been achieved by cross-polarization, and high-resolution 1H spectra have been recorded using the wPMLG3 decoupling during the t 2 evolution period. The performance of the pulse sequences have been tested on the templated microporous aluminophosphate IST-1 and trisodium citrate employing a B 0 field of 9.4 T.

Proton-proton homonuclear dipolar decoupling in solid-state NMR using rotor-synchronized z-rotation pulse sequences

We present a theoretical analysis of rotor-synchronized homonuclear dipolar decoupling schemes that cause a z-rotation of the spins. These pulse sequences applicable at high spinning rates (nu(r) > or = 30 kHz) yield high-resolution proton NMR spectra that are free of artifacts, such as zero lines and image peaks. We show that the scaled isotropic chemical-shift positions of proton lines can be calculated from the zero-order average Hamiltonian and that the scaling factor does not depend on offset. The effects of different adjustable parameters (rf field, spinning rate, pulse shape, offset) on the decoupling performance are analyzed by numerical simulations of proton spectra and by (1)H solid-state NMR experiments on NaH(2)PO(4) and glycine.

High-Resolution Solution NMR Spectra in Inhomogeneous Magnetic Fields

A high-resolution NMR spectrum, containing fine structures such as chemical shifts and scalar-coupling multiplet patterns, is helpful for analyzing molecular composition and structure. However, there are many cases where the magnetic environments are inhomogeneous, which may lead to severe signal overlapping and blur useful spectral information in liquid samples. Recently, many NMR techniques have been proposed to regain high-resolution spectral information in inhomogeneous magnetic fields. They can be mainly classified into three types that based on intermolecular multiple- quantum coherences, nutation echoes, and spatial encoding respectively. These methods can be applied to a wide range of samples, thereby opening a way to high-resolution in vivo and ex situ NMR spectroscopy. In this review, recent developments of these methods are presented and their applicability and efficiency are analyzed.

Spin-coated and PECVD low dielectric constant porous organosilicate films studied by 1D and 2D solid-state NMR

In the research field of the sub-65 nm semiconductor industry, organosilicate SiOCH films with low dielectric constant (k < 2.4) need to be developed in order to improve the performance of integrated circuits [International Roadmap for Semiconductors (ITRS), San Jose, CA, 2004]. One way to produce SiOCH films of low dielectric constant is to introduce pores into the film. This is usually obtained in two steps. Firstly, co-deposition of a matrix precursor, with a sacrificial organic porogen, either by plasma enhanced chemical vapor deposition (PECVD) or spin-coating. Secondly, application of a specific thermal treatment to remove the porogen and create the porosity. This last step can be improved by adding to the thermal process a super-critical CO(2) treatment, an UV irradiation or an electronic bombardment (e-beam). In this study, the two deposition processes as well as the various treatments applied to eliminate the porogens were evaluated and compared using high-resolution solid-state NMR. For this purpose, hybrid (containing porogens) and porous films were extensively characterized on the basis of their (1)H, (13)C and (29)Si high-resolution NMR spectra. Information was obtained concerning the crosslinking of the Si skeleton. Spectral features could be correlated to the processes used. Isotropic chemical shift analyses and 2D correlation NMR experiments were used to show the existence and nature of the interactions between the matrix precursor and the organic porogen.

The unambiguous assignment of NMR spectra of per-O-methylated 6-mono and 6,6-diamino-β-cyclodextrins

The step-by-step analysis of high-resolution proton and 13C NMR spectra of per-O-methylated-6-monoamino and 6,6-diamino-A,D-β-cyclodextrins is described. Selected 2-D experiments (COSY, NOESY, HSQC, DEPT, and HMBC) allowed us to challenge the current interpretation. The full assignment of the proton and carbon-13 NMR spectra of these two compounds was performed enabling us to complete the assignment of NMR spectra acquired at 500 and 800 MHz for these important synthetic intermediates.Key words: COSY, NOESY, HSQC, DEPT, HMBC, NMR, 6-amino-β-cyclodextrins, 6,6-diamino-β-cyclodextrins.

High-resolution NMR spectra in inhomogeneous fields utilizing the CRAZED sequence without coherence selection gradients

Coherence selection gradients have been considered as indispensable for high-resolution NMR spectroscopy in inhomogeneous fields utilizing the CRAZED-type sequences. However, our experimental results demonstrate that these gradients can be omitted if an appropriate phase cycling is applied. The measured linewidth of reconstructing 1D high-resolution spectral peaks does not depend on the dipolar correlation distance determined by the coherence selection gradients, but is only affected by diffusion and T 2 relaxation. This finding suggests the need to reconsider the mechanism for the iMQC-based high-resolution spectroscopy.