The effectiveness of 1H decoupling in the 13C MAS NMR of paramagnetic solids: an experimental case study incorporating copper(II) amino acid complexes

The use of continuous-wave (CW) 1H decoupling has generally provided little improvement in the 13C MAS NMR spectroscopy of paramagnetic organic solids. Recent solid-state 13C NMR studies have demonstrated that at rapid magic-angle spinning rates CW decoupling can result in reductions in signal-to-noise and that 1H decoupling should be omitted when acquiring 13C MAS NMR spectra of paramagnetic solids. However, studies of the effectiveness of modern 1H decoupling sequences are lacking, and the performance of such sequences over a variety of experimental conditions must be investigated before 1H decoupling is discounted altogether. We have studied the performance of several commonly used advanced decoupling pulse sequences, namely the TPPM, SPINAL-64, XiX, and eDROOPY sequences, in 13C MAS NMR experiments performed under four combinations of the magnetic field strength (7.05 or 11.75T), rotor frequency (15 or 30kHz), and 1H rf-field strength (71, 100, or 140kHz). The effectiveness of these sequences has been evaluated by comparing the 13C signal intensity, linewidth at half-height, LWHH, and coherence lifetimes, T2('), of the methine carbon of copper(II) bis(dl-alanine) monohydrate, Cu(ala)(2).H2O, and methylene carbon of copper(II) bis(dl-2-aminobutyrate), Cu(ambut)(2), obtained with the advanced sequences to those obtained without 1H decoupling, with CW decoupling, and for fully deuterium labelled samples. The latter have been used as model compounds with perfect 1H decoupling and provide a measure of the efficiency of the 1H decoupling sequence. Overall, the effectiveness of 1H decoupling depends strongly on the decoupling sequence utilized, the experimental conditions and the sample studied. Of the decoupling sequences studied, the XiX sequence consistently yielded the best results, although any of the advanced decoupling sequences strongly outperformed the CW sequence and provided improvements over no 1H decoupling. Experiments performed at 7.05T demonstrate that the XiX decoupling sequence is the least sensitive to changes in the 1H transmitter frequency and may explain the superior performance of this decoupling sequence. Overall, the most important factor in the effectiveness of 1H decoupling was the carbon type studied, with the methylene carbon of Cu(ambut)(2) being substantially more sensitive to 1H decoupling than the methine carbon of Cu(ala)(2).H2O. An analysis of the various broadening mechanisms contributing to 13C linewidths has been performed in order to rationalize the different sensitivities of the two carbon sites under the four experimental conditions.     

REDOR-determined distances from heterospins to clusters of 13C labels

The use of rotational-echo double resonance NMR to measure distances from an observed tightly coupled cluster of 13C spins to a distant 15N, 31P, or 19F is practical if all homonuclear 13C-13C dipolar interactions are suppressed by multiple-pulse decoupling during heteronuclear dipolar evolution. This scheme is first calibrated by experiments performed on multiply labeled alanines and then applied in the measurement of 19F-13C distances in p-trifluoromethylphenyl [1,2-13C2]acetate.     

SPINAL modulated decoupling in high field double- and triple-resonance solid-state NMR experiments on stationary samples

Continuous wave irradiation has limited bandwidth for heteronuclear 1H decoupling at high fields and for 13C decoupling in 1H/13C/15N triple-resonance experiments. SPINAL-16 modulation is shown to improve the efficiency of 1H and 13C heteronuclear decoupling on single crystals of peptides and on magnetically aligned samples of membrane proteins in bicelles, which is of particular importance because aqueous samples of biomolecules are lossy at high fields, which limits the strengths of the RF fields that can be applied.     

Interference of homonuclear decoupling and exchange in the solid-state NMR of perfluorocyclohexane

We observe an interference between RF irradiation used for homonuclear decoupling of 19F and conformational exchange in the 13C spectrum of perfluorocyclohexane. We show that these effects can be readily reproduced in simulation, and characterise their dependence on the various NMR and experimental parameters. Their application to observing exchange rates on the kHz timescale is evaluated with respect to T(1rho) measurements and the connections between the two approaches established. The implications for experiments that use homonuclear decoupling of 1H to resolve 1J(CH)couplings in the solid-state are also evaluated in detail.     

Dipole-coupled carbon-13 spectra,a source of structural information on liquid crystals

It is shown that the introduction of well-selected dipolar interactions into carbon-13 spin systems of liquid crystals can lead to well-resolved spectra with a rich content of structural information. Two possibilities are investigated: (a) selective isotope substitution, in particular by deuterium and nitrogen-15, and simultaneous proton decoupling; (b) two-dimensional separated local field carbon-13 spectroscopy. The methods are demonstrated by investigations of benzylidene anilines in their nematic phases.     

Background signal in solid state 13C NMR spectra of soil organic matter (SOM)-quantification and minimization

13C background signal, obtained for an empty rotor, was shown by spin counting experiments to be equivalent to 1 mg of observable carbon for cross-polarization (CP) spectra and 69 mg of observable C for Bloch decay (BD) spectra. The BD background was mainly due to Kel-F in the stator. with minimal signal detected from the Kel-F end-caps. The CP background was attributed to non-Kel-F components of the stator, probe, or probe supports. The BD background signal was eliminated by using a modified dipolar dephasing pulse sequence in which the absence of 19F decoupling (rather than the absence of 1H decoupling) causes selective elimination of the Kel-F signal.     

Suppression of artifacts induced by homonuclear decoupling in amino-acid-type edited methyl 1H-13C correlation experiments

A detailed theoretical and experimental analysis of the artifacts induced by homonuclear band-selective decoupling during CT frequency labeling is presented. The effects are discussed in the context of an amino-acid-type editing filter implemented in (1)H-(13)C CT-HSQC experiments of methyl groups in proteins. It is shown that both Bloch-Siegert shifts and modulation sidebands are efficiently suppressed by using additional off-resonance decoupling as proposed by Zhang and Gorenstein [J. Magn. Reson. 132 (1998) 81], and appropriate adjustment of a set of pulse sequence parameters. The theoretical predictions are confirmed by experiments performed on (13)C-labeled protein samples, yielding artifact-free amino-acid-type edited methyl spectra.     

Measurement of the principal values of the anisotropic diffusion tensor in an unoriented sample by exploiting the chemical shift anisotropy: (19)F PGSE NMR with homonuclear decoupling

NMR methods (S. V. Dvinskikh et al., J. Magn. Reson. 142, 102-110 (2000) and S. V. Dvinskikh and I. Furó, J. Magn. Reson. 144, 142-149 (2000)) that combine PGSE with dipolar decoupling are extended to polycrystalline solids and unoriented liquid crystals. Decoupling suppresses dipolar dephasing not only during the gradient pulses but also under signal acquisition so that the detected spectral shape is dominated by the chemical shift tensor of the selected nucleus. The decay of the spectral intensity at different positions in the powder spectrum provides the diffusion coefficient in sample regions with their crystal axes oriented differently with respect to the direction of the field gradient. Hence, one can obtain the principal values of the diffusion tensor. The method is demonstrated by (19)F PGSE NMR with homonuclear decoupling in a lyotropic lamellar liquid crystal.     

Carbon-13 and fluorine-19 NMR spectroscopy of the supramolecular solid p-tert-butylcalix(4)arene.alpha,alpha,alpha-trifluorotoluene

The supramolecular 1:1 host-guest inclusion compound, p-tert-butylcalix[4]arene x alpha,alpha,alpha-trifluorotoluene, 1, is characterized by 19F and 13C solid-state NMR spectroscopy. Whereas the 13C NMR spectra are easily interpreted in the context of earlier work on similar host-guest compounds, the 15F NMR spectra of solid 1 are, initially, more difficult to understand. The 19F[1H] NMR spectrum obtained under cross-polarization and magic-angle spinning conditions shows a single isotropic resonance with a significant spinning sideband manifold. The static 19F[1H] CP NMR spectrum consists of a powder pattern dominated by the contributions of the anisotropic chemical shift and the homonuclear dipolar interactions. The 19F MREV-8 experiment, which minimizes the 19F-19F dipolar contribution, helps to identify the chemical shift contribution as an axial lineshape. The full static 19F[1H] CP NMR spectrum is analysed using subspectral analysis and subsequently simulated as a function of the 19F-19F internuclear distance (D(FF) = 2.25 +/- 0.01 A) of the rapidly rotating CF3 group without including contributions from additional libration motions and the anisotropy in the scalar tensor. The shielding span is found to be 56 ppm. The width of the centerband in the 19F[1H] sample-spinning CP NMR spectrum is very sensitive to the angle between the rotor and the magnetic field. Compound 1 is thus an attractive standard for setting the magic angle for NMR probes containing a fluorine channel with a proton-decoupling facility.     

13C PGSE NMR experiment with heteronuclear dipolar decoupling to measure diffusion in liquid crystals and solids

A new PGSE NMR experiment, designed to measure molecular diffusion coefficients in systems with nonvanishing static dipolar coupling, is described. The fast static dipolar dephasing of the single-quantum (13)C coherences is removed by multiple-pulse heteronuclear decoupling. The resulting slow dephasing of the (13)C coherences allows for inserting appropriate gradient pulses into the pulse sequence. The presence of the large magnetic field gradient reduces the efficiency of the decoupling sequences which is compensated for by introducing a scheme of sequential slice selection across the sample. The method is demonstrated by (19)F-decoupled (13)C PGSE NMR experiments in a lyotropic nematic and lamellar liquid crystal.     

Band-selective recoupling of homonuclear double-quantum dipolar interaction with a generalized composite 0 degrees pulse: application to 13C aliphatic region-selective magnetization transfer in solids

Recoupling of homonuclear double quantum (DQ)-dipolar interactions is a useful technique for the structural analysis of molecules in solids. We have designed a series of elemental 0 degrees pulses for the recoupling sequences with the rf phase rotation about the z-axis, known as CN. The proposed 0 degrees pulses whose total flip angle >/=360 degrees provide spin rotation vectors in the xy-plane. Thus, the residual spin rotation can be canceled by rf phase rotation about the z-axis. An analysis by the coherent averaging theory showed that effective bandwidths of the recoupling sequences are limited not by the reduction in the dipolar scaling factor but by the increase in the residual spin rotation due to offset. A CN sequence with these elemental pulses provides an effective bandwidth of DQ-dipolar recoupling from ca. 0.5nu(R) to 4nu(R) for numerical simulations. Here, nu(R) is the sample spinning frequency. The 0 degrees pulses were applied to band-selective recoupling for the magnetization transfer in uniformly 13C-labeled molecules. Narrow-band recoupling enhances the magnetization transfer between spins within the effective range by decoupling the dipolar interactions between spins one of which is outside the range. The narrow band operation reduces rf field strength, which improves the CH decoupling. Increases in signal intensities by the use of the proposed 0 degrees pulses are experimentally shown for 13C-labeled amino acids.     

Resolution enhancement in spectra of natural products dissolved in weakly orienting media with the help of 1H homonuclear dipolar decoupling during acquisition: application to 1H-13C dipolar couplings measurements

In weakly orienting media such as poly-gamma-benzyl-L-glutamate (PBLG) a polymer that forms a chiral liquid crystal in organic solvents, the spectral resolution for embedded molecules is usually poor because of numerous (1)H, (1)H dipolar couplings that generally broaden proton spectra. Therefore (1)H, (13)C dipolar couplings are difficult or impossible to measure. Here, we incorporate Flip-Flop decoupling during detection into an HSQC experiment. Flip-Flop removes the (1)H, (1)H dipolar couplings and scales the chemical shifts of the protons as well as the (1)H, (13)C dipolar couplings during detection. A resolution gain by a factor 1.5-4.2 and improved signal intensity by an average factor of 1.6-1.7 have been obtained. This technique is demonstrated on (+)-menthol dissolved in a PBLG/CDCl(3) phase.     

13C-1H HSQC experiment of probe molecules aligned in thermotropic liquid crystals: sensitivity and resolution enhancement in the indirect dimension

The spectra of molecules oriented in liquid crystalline media are dominated by partially averaged dipolar couplings. In the 13C-1H HSQC, due to the inefficient hetero-nuclear dipolar decoupling in the indirect dimension, normally carried out by using a pi pulse, there is a considerable loss of resolution. Furthermore, in such strongly orienting media the 1H-1H and 13C-1H dipolar couplings leads to fast dephasing of transverse magnetization causing inefficient polarization transfer and hence the loss of sensitivity in the indirect dimension. In this study we have carried out 13C-1H HSQC experiment with efficient polarization transfer from 1H to 13C for molecules aligned in liquid crystalline media. The homonuclear dipolar decoupling using FFLG during the INEPT transfer delays and also during evolution period combined with the pi pulse heteronuclear decoupling in the t1 period has been applied. The studies showed a significant reduction in partially averaged dipolar couplings and thereby enhancement in the resolution and sensitivity in the indirect dimension. This has been demonstrated on pyridazine and pyrimidine oriented in the liquid crystal. The two closely resonating carbons in pyrimidine are better resolved in the present study compared to the earlier work [H.S. Vinay Deepak, Anu Joy, N. Suryaprakash, Determination of natural abundance 15N-1H and 13C-1H dipolar couplings of molecules in a strongly orienting media using two-dimensional inverse experiments, Magn. Reson. Chem. 44 (2006) 553-565].     

An improved broadband decoupling sequence for liquid crystals and solids

Recently we developed an efficient broadband decoupling sequence called SPARC-16 for liquid crystals ?J. Magn. Reson. 130, 317 (1998). The sequence is based upon a 16-step phase cycling of the 2-step TPPM decoupling method for solids ?J. Chem. Phys. 103, 6951 (1995). Since then, we have found that a stepwise variation of the phase angle in the TPPM sequence offers even better results. The application of this new method to a liquid crystalline compound, 4-n-pentyl-4'-cyanobiphenyl, and a solid, L-tyrosine hydrochloride, is reported. The reason for the improvement is explained by an analysis of the problem in the rotating frame.     

Rotational-echo double resonance of uniformly labeled 13C clusters

The use of rotational-echo double resonance NMR to measure distances from an observed tightly coupled cluster of 13C spins to a distant 15N, 31P, or 19F is practical if 13C chemical shifts and homonuclear 13C-13C isotropic J interactions are refocused by a combination of rotor-synchronized 13C pi and pi/2 pulses. This scheme is illustrated by experiments performed on diluted and recrystallized L-[13C(3),15N]alanine and L-[13C(6),alpha-15N]histidine.     

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.     

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.     

An NMR study of the origin of dioxygen-induced spin-lattice relaxation enhancement and chemical shift perturbation

Due to its depth-dependent solubility, oxygen exerts paramagnetic effects which become progressively greater toward the hydrophobic interior of micelles, and lipid bilayer membranes. This paramagnetic gradient, which is manifested as contact shift perturbations (19F and 13C NMR) and spin-lattice relaxation enhancement (19F and 1H NMR), has been shown to be useful for precisely determining immersion depth, membrane protein secondary structure, and overall topology of membrane proteins. We have investigated the influence of oxygen on 19F and 13C NMR spectra and spin-lattice relaxation rates of a semiperfluorinated detergent, (8,8,8)-trifluoro (3,3,4,4,5,5,6,6,7,7)-difluoro octylmaltoside (TFOM) in a model membrane system, to determine the dominant paramagnetic spin-lattice relaxation and shift-perturbation mechanism. Based on the ratio of paramagnetic spin-lattice relaxation rates of 19F and directly bonded 13C nuclei, we conclude that the dominant relaxation mechanism must be dipolar. Furthermore, the temperature dependence of oxygen-induced chemical shift perturbations in 9F NMR spectra suggests a contact interaction is the dominant shift mechanism. The respective hyperfine coupling constants for 19F and 13C nuclei can then be estimated from the contact shifts <(deltav/v0)19F> and <(deltav/v0)13C>, allowing us to estimate the relative contribution of scalar and dipolar relaxation to 19F and 13C nuclei. We conclude that the contribution to spin-lattice relaxation from the oxygen induced paramagnetic scalar mechanism is negligible.     

Selective saturation and inversion of multiple resonances in high-resolution solid-state 13C experiments using slow spinning CP/MAS and tailored DANTE pulse sequences.

Taking advantage of the long 13C T1 values generally encountered in solids, selective saturation and inversion of more than one resonance in 13C CP/MAS experiments can be achieved by sequentially applying several DANTE pulse sequences centered at different transmitter frequency offsets. A new selective saturation pulse sequence is introduced composed of a series of 90 degrees DANTE sequences separated by interrupted decoupling periods during which the selected resonance is destroyed. Applications of this method, including the simplification of the measurement of the principal values of the 13C chemical shift tensor under slow MAS conditions, are described. The determination of the aromaticity of coal using a relatively slow MAS rate is also described.     

氨基酸合成方法研究—(XI)N-取代亚氨酸酯与芳醛缩合产物—去氢氨基酸衍生物的构型及其NMR研究

通过~1H NMR、NOE差谱和门控去偶~(13)C NMR等的研究确定了新合成的五对α-去氢氨基酸衍生物的构型,并利用部分弛豫、APT~(13)C NMR二维碳氢相关谱及~(13)C-~(19)F偶合常数等对NMR谱线进行了归属.