Improvement of resolution in solid state NMR spectra with J-decoupling: an analysis of lineshape contributions in uniformly 13C-enriched amino acids and proteins

In magic angle spinning (MAS) NMR spectra of highly and uniformly 13C,15N-enriched amino acids and proteins, homo-nuclear coupling interactions contribute significantly to the 13C linewidths, particularly for moderate applied magnetic field strengths and sample spinning frequencies. In this work, we attempted to dissect, analyze, and control the contributions of J-coupling and residual homo-nuclear dipolar coupling interactions to the linewidths of uniformly 13C,15N-enriched crystalline alanine; these studies were carried out at 9.4 T using a range of spinning frequencies from 5 to 15 kHz. The anisotropic second-order dipolar shifts and the J-splittings are comparable in their contribution to the linewidths, but behave very differently in terms of experimental protocols for line narrowing. In contrast to the J-coupling interactions, the second-order dipolar broadening cannot be refocused using selective pulses on the passively coupled spin. We carried out experiments to remove or refocus the 13C J-coupling interactions (omega1 J-decoupling) using a selective DANTE pulse in the center of the indirect evolution period. Inversion profiles and bandwidths of selective DANTE pulses acting on transverse magnetization, in the regime of moderate spinning frequencies, were characterized computationally and experimentally. A dramatic improvement in the resolution of the 2D spectrum was achieved when this decoupling protocol was employed.     

Rotational resonance in uniformly 13C-labeled solids: effects on high-resolution magic-angle spinning NMR spectra and applications in structural studies of biomolecular systems

We describe investigations of the effects of rotational resonance (R(2)) on solid state (13)C NMR spectra of uniformly (13)C-labeled samples obtained under magic-angle spinning (MAS), and of the utility of R(2) measurements as structural probes of peptides and proteins with multiple uniformly labeled residues. We report results for uniformly (13)C-labeled L-alanine and L-valine in polycrystalline form, and for amyloid fibrils formed by the 15-residue peptide A beta(11-25) with uniform labeling of a four-residue segment. The MAS NMR spectra reveal a novel J-decoupling effect at R(2) conditions that may be useful in spectral assignments for systems with sharp (13)C MAS NMR lines. Pronounced dependences of the apparent isotropic (13)C NMR chemical shifts on MAS frequency near R(2) conditions are also observed. We demonstrate the feasibility of quantitative (13)C-(13)C distance determinations in L-valine, and qualitative determinations of inter-residue (13)C-(13)C contacts in A beta(11-25) fibrils. Finally, we demonstrate a "relayed" R(2) technique that may be useful in structural measurements on systems with poorly resolved (13)C MAS NMR lines.     

13C high-resolution NMR study of doped polyacetylene

¹³C high resolution NMR spectra of doped trans polyacetylene, (CH)_x, have been measured. A spectrum of (CH)_x doped with AsF₅ shows a downfield shift, while that with potassium an upfield shift. The charge distribution and the existence of sp³ hybridized carbon on halogen-doped (CH)_x chains are discussed.     

Improvement of resolution in solid state NMR spectra with J-decoupling: an analysis of lineshape contributions in uniformly C-enriched amino acids and proteins

In magic angle spinning (MAS) NMR spectra of highly and uniformly 13C,15N-enriched amino acids and proteins, homo-nuclear coupling interactions contribute significantly to the 13C linewidths, particularly for moderate applied magnetic field strengths and sample spinning frequencies. In this work, we attempted to dissect, analyze, and control the contributions of J-coupling and residual homo-nuclear dipolar coupling interactions to the linewidths of uniformly 13C,15N-enriched crystalline alanine; these studies were carried out at 9.4 T using a range of spinning frequencies from 5 to 15 kHz. The anisotropic second-order dipolar shifts and the J-splittings are comparable in their contribution to the linewidths, but behave very differently in terms of experimental protocols for line narrowing. In contrast to the J-coupling interactions, the second-order dipolar broadening cannot be refocused using selective pulses on the passively coupled spin. We carried out experiments to remove or refocus the 13C J-coupling interactions (omega1 J-decoupling) using a selective DANTE pulse in the center of the indirect evolution period. Inversion profiles and bandwidths of selective DANTE pulses acting on transverse magnetization, in the regime of moderate spinning frequencies, were characterized computationally and experimentally. A dramatic improvement in the resolution of the 2D spectrum was achieved when this decoupling protocol was employed.     

Magnetic susceptibility effects on 13C MAS NMR spectra of carbon materials and graphite

13C high-resolution solid-state nuclear magnetic resonance (NMR) was employed to study carbon materials prepared through the thermal decomposition of four different organic precursors (rice hulls, endocarp of babassu coconut, peat, and PVC). For heat treatment temperatures (HTTs) above about 600 C, all materials presented 13C NMR spectra composed of a unique resonance line associated with carbon atoms in aromatic planes. With increasing HTT a continuous broadening of this resonance and a diamagnetic shift in its central frequency were verified for all samples. The evolution of the magnitude and anisotropy of the magnetic susceptibility of the heat-treated carbon samples with HTT explains well these findings. It is shown that these results are better understood when a comparison is made with the features of the 13C NMR spectrum of polycrystalline graphite, for which the magnetic susceptibility effect is also present and is much more pronounced.     

Aromatic spectral editing techniques for magic-angle-spinning solid-state NMR spectroscopy of uniformly 13C-labeled proteins

The four aromatic amino acids in proteins, namely histidine, phenylalanine, tyrosine, and tryptophan, have strongly overlapping ¹³C chemical shift ranges between 100 and 160 ppm, and have so far been largely neglected in solid-state NMR determination of protein structures. Yet aromatic residues play important roles in biology through π–π and cation–π interactions. To better resolve and assign aromatic residues' ¹³C signals in magic-angle-spinning (MAS) solid-state NMR spectra, we introduce two spectral editing techniques. The first method uses gated ¹H decoupling in a proton-driven spin-diffusion (PDSD) experiment to remove all protonated ¹³C signals and retain only non-protonated carbon signals in the aromatic region of the ¹³C spectra. The second technique uses chemical shift filters and ¹H–¹³C dipolar dephasing to selectively detect the Cα, Cβ and CO cross peaks of aromatic residues while suppressing the signals of all aliphatic residues. We demonstrate these two techniques on amino acids, a model peptide, and the microcrystalline protein GB1, and show that they significantly simplify the 2D NMR spectra and both reveal and permit the ready assignment of the aromatic residues' signals.     

13C NMR chemical shift of single-wall carbon nanotubes

We compute the magnetic shielding tensor within the London approximation and estimate the Knight shift of single-wall carbon nanotubes. Our results indicate that high resolution 13C NMR should be able to separate the metallic and insulator character of the nanotubes since a 11 ppm splitting is predicted from the respective resonances. As a model for disorder, bending, and defects in these structures, we investigate the magnetic response of nanotubes with finite size. We get a small line broadening coming from an intrinsic length dependent resonance effect. The nanotube packing is also studied and leads to a 20 ppm broadening which disappears under experimental high-resolution conditions.     

Three-dimensional 13C shift/1H-15N coupling/15N shift solid-state NMR correlation spectroscopy.

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly 13C- and 15N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from 1H to 13C and from 13C to 15N with flip-flop (phase and frequency switched) Lee-Goldburg irradiation for both 13C homonuclear decoupling and 1H-15N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields 13C shift/1H-15N coupling/15N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate 13C and 15N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly 13C- and 15N-labeled proteins.     

Inhomogeneity-free heteronuclear iMQC

Intermolecular dipolar interactions between proton and carbon spins can be used to indirectly detect carbon spectra with high sensitivity. In this communication, we present a modified sequence that, in addition to the high sensitivity of heteronuclear intermolecular multiple quantum coherence (iMQC) experiments, retains the line narrowing capability characteristic of homonuclear zero-quantum coherences. We demonstrate that this sequence can be used to obtain high resolution (13)C spectra in the presence of magnetic field inhomogeneities, both for thermal and hyperpolarized samples, and discuss applications to water-hyperpolarized carbon imaging.     

A 13C solid-state NMR investigation of the alkynyl carbon chemical shift tensors for 2-butyne-1,4-diol

The alkynyl carbon chemical shift (CS) tensors for 2-butyne-1,4-diol are reported, based on analyses of the carbon-13 NMR spectra of stationary-powder and slow magic-angle spinning (MAS) samples for which the alkynyl carbon nuclei are enriched in 13C. NMR spectra of slow MAS samples exhibit spinning-frequency-dependent fine structure typical of crystallographically equivalent but magnetically distinct nuclei. Simulated spectra of slow MAS samples of this two-spin system are particularly sensitive to the relative orientations of the CS tensors. In addition, the value of 1J(13C, 13C), +175 +/- 10 Hz, is determined by examination of the total NMR lineshape of slow MAS samples. The CS tensors are almost axially symmetric, delta11 = 158.9 +/- 1.0 ppm and delta22 = 155.7 +/- 1.0 ppm; the direction of greatest shielding is approximately along the alkynyl C-C bond, delta33 = -57.8 +/- 2.0 ppm. Both the magnitudes of the principal components of the CS tensors and their orientations are in agreement with those predicted from first-principles calculations at the HF and MP2 levels of theory. This study demonstrates the importance of examining the NMR spectra of homonuclear two-spin systems with and without MAS under a variety of conditions (e.g., two or more applied magnetic fields and slow MAS).     

吉士脱酮的1H及13C NMR研究

Gestodene是避孕药物18甲基炔诺酮的衍生物,动物实验表明它的孕激素活性为18甲基炔诺酮的3~5倍.本文采用各种2D NMR技术,包括:¹H-¹H COSY,¹H-¹³C COSY和HMBC等,归属了它的¹H和¹³C的谱线,并得到了有关质子间的偶合常数.     

Compressed sensing for resolution enhancement of hyperpolarized 13C flyback 3D-MRSI

High polarization of nuclear spins in liquid state through dynamic nuclear polarization has enabled the direct monitoring of 13C metabolites in vivo at very high signal-to-noise, allowing for rapid assessment of tissue metabolism. The abundant SNR afforded by this hyperpolarization technique makes high-resolution 13C 3D-MRSI feasible. However, the number of phase encodes that can be fit into the short acquisition time for hyperpolarized imaging limits spatial coverage and resolution. To take advantage of the high SNR available from hyperpolarization, we have applied compressed sensing to achieve a factor of 2 enhancement in spatial resolution without increasing acquisition time or decreasing coverage. In this paper, the design and testing of compressed sensing suited for a flyback 13C 3D-MRSI sequence are presented. The key to this design was the undersampling of spectral k-space using a novel blipped scheme, thus taking advantage of the considerable sparsity in typical hyperpolarized 13C spectra. Phantom tests validated the accuracy of the compressed sensing approach and initial mouse experiments demonstrated in vivo feasibility.     

Two-dimensional dynamic-director (13)C NMR of liquid crystals

A novel nuclear magnetic resonance (NMR) experiment for facilitating the resolution and assignment of liquid crystalline (13)C NMR spectra is described. The method involves the motor-driven reorientation of the liquid crystalline director, in synchrony with the acquisition of a 2D chemical shift correlation spectrum. By monitoring in this fashion the (13)C NMR evolution of spins in the liquid crystal at two different director orientations with respect to the magnetic field, the method distinguishes anisotropic from isotropic displacements and can be utilized for assigning the resonances and estimating local degrees of order. Of various potential pairs of angles suitable for such a correlation, the (0 degrees, 90 degrees ) choice was found to be most convenient, as it avoids line broadening complications that may otherwise originate from heterogeneities of the oriented phase. The technique thus derived was employed in the analysis of a series of monomeric and polymeric liquid crystal systems.     

A supplementary coil for ²H decoupling with commercial HCN MAS probes

Partial deuteration is a powerful tool to increase coherence life times and spectral resolution in proton solid-state NMR. The J coupling to deuterium needs, however, to be decoupled to maintain the good resolution in the (usually indirect) (13)C dimension(s). We present a simple and reversible way to expand a commercial 1.3mm HCN MAS probe with a (2)H channel with sufficient field strength for J-decoupling of deuterium, namely 2-3kHz. The coil is placed at the outside of the stator and requires no significant modifications to the probe. The performance and the realizable gains in sensitivity and resolution are demonstrated using perdeuterated ubiquitin, with selectively CHD(2)-labeled methyl groups.     

Two-dimensional high-resolution NMR spectra in matched B(0) and B(1) field gradients

In a recent publication we presented a method to obtain highly resolved NMR spectra in the presence of an inhomogeneous B(0) field with the help of a matched RF gradient. If RF gradient pulses are combined with "ideal" 90 degrees pulses to form inhomogeneous z rotation pulses, the line broadening caused by the B(0) gradient can be refocused, while the full chemical shift information is maintained. This approach is of potential use for NMR spectroscopy in an inhomogeneous magnetic field produced by an "ex-situ" surface spectrometer. In this contribution, we extend this method toward two-dimensional spectroscopy with high resolution in one or both dimensions. Line narrowing in the indirect dimension can be achieved by two types of nutation echoes, thus leading to depth-sensitive NMR spectra with full chemical shift information. If the nutation echo in the indirect dimension is combined with a stroboscopic acquisition using inhomogeneous z-rotation pulses, highly resolved two-dimensional correlation spectra can be obtained in matched field gradients. Finally, we demonstrate that an INEPT coherence transfer from proton to carbon spins is possible in inhomogeneous B(0) fields. Thus, it is possible to obtain one-dimensional (13)C NMR spectra with increased sensitivity and two-dimensional HETCOR spectra in the presence of B(0) gradients of 0.4 mT/cm. These schemes may be of some value for ex-situ NMR analysis of materials and biological systems.     

Asymmetric MRI magnet design using a hybrid numerical method

Two-dimensional 1H/13C polarization inversion spin exchange at the magic angle experiments were applied to single crystal samples of amino acids to demonstrate their potential utility on oriented samples of peptides and proteins. High resolution is achieved and structural information obtained on backbone and side chain sites from these spectra. A triple-resonance experiment that correlates the 1H-13Calpha dipolar coupling frequency with the chemical shift frequencies of the alpha-carbon, as well as the directly bonded amide 15N site, is also demonstrated. In this experiment the large 1H-13Calpha heteronuclear dipolar interaction provides an independent frequency dimension that significantly improves the resolution among overlapping 13C resonances of oriented polypeptides, while simultaneously providing measurements of the 13Calpha chemical shift, 1H-13C dipolar coupling, and 15N chemical shift frequencies and angular restraints for backbone structure determination.     

13C NMR Studies on 3-Aryl-4-Cyanosydnones (II). NMR Spectroscopy and the Chain-Conjugated Structure of Sydnones

Carbon-13 NMR spectra of 3-aryl-4-cyanosydnones have been analyzed to serve as an electronic structure probe of sydnones. 2-Dimensional INADEQUATE experiments and spin-lattice relaxation time measurements were employed to ascertain exact assignments. Deshielding of the phenyl carbon para to the sydnone ring manifests that N(3) bears positive charge. The electronic-rich property of C(4) is confirmed by shift data of cyano carbons, which have been found by far the most shielded cyano carbons in nitrile-containing compounds yet reported. Shift variations of C(5) resulting from the substitution at C(4) and supplementary oxygen-17 chemical shift data provide evidence in favor of the chain-conjugated structure.     

Pursuing structure in microcrystalline solids with independent molecules in the unit cell using 1H-13C correlation data

The (1)H-(13)C solid-state NMR heteronuclear correlation (HETCOR) experiment is demonstrated to provide shift assignments in certain powders that have two or more structurally independent molecules in the unit cell (i.e. multiple molecules per asymmetric unit). Although this class of solids is often difficult to characterize using other methods, HETCOR provides both the conventional assignment of shifts to molecular positions and associates many resonances with specific molecules in the asymmetric unit. Such assignments facilitate conformational characterization of the individual molecules of the asymmetric unit and the first such characterization solely from solid-state NMR data is described. HETCOR offers advantages in sensitivity over prior methods that assign resonances in the asymmetric unit by (13)C-(13)C correlations and therefore allows shorter average analysis times in natural abundance materials. The (1)H-(13)C analysis is demonstrated first on materials with known shift assignments from INADEQUATE data (santonin and Ca(OAc)(2) phase I) to verify the technique and subsequently is extended to a pair of unknown solids: (+)-catechin and Ca(OAc)(2) phase II. Sufficient sensitivity and resolution is achieved in the spectra to provide assignments to one of the specific molecules of the asymmetric unit at over 54% of the sites.     

On the 13C CP/MAS spectra of leucine.

The CP/MAS 13C NMR spectra of crystalline L-leucine and DL-leucine at 7 T are compared with previously reported spectra at lower field strengths. An increasing dominance of chemical shift effects over residual 14N-13C dipolar interactions is observed on the C alpha and C beta splittings with increasing field strength. A new structure is observed in the 25 ppm region of both samples. The spectra in this region were assigned by application of the depolarisation-repolarisation method. The assignment showed differences in the ordering of peaks between solid state and liquid state chemical shifts.     

Fourier Transform 13C NMR Analysis of Some Methaqualone Metabolites

The natural abundance carbon-13 nuclear magnetic resonance spectra of some methaqualone metabolites were recorded using the pulse fourier transform technique. The chemical shift of various carbon resonances have been assigned on the basis of the chemical shift theory, multiplicities observed in SFORD spectrum and comparison with the chemical shifts of the corresponding carbons of methaqualone.