13C/14N heteronuclear multiple-quantum correlation with rotary resonance and REDOR dipolar recoupling

A two-dimensional (13)C/(14)N heteronuclear multiple quantum correlation (HMQC) experiment using dipolar recoupling under magic-angle spinning (MAS) is described. The experiment is an extension of the recent indirect (13)C detection scheme for measuring (14)N quadrupolar coupling under MAS. The recoupling allows the direct use of the much larger dipolar interaction instead of the small J and residual dipolar couplings for establishing (13)C/(14)N correlations. Two recoupling methods are incorporated into the HMQC sequence, both applying rf only to the observed (13)C spin. The first one uses the REDOR sequence with two pi-pulses per rotor cycle. The second one uses a cw rf field matching the spinning frequency, known as rotary resonance. The effects of CSA, T(2)(') signal loss, MAS frequency and stability and t(1)-noise are compared and discussed.     

A 4-mm probe for (13)C CP/MAS NMR of solids at 21.15 T

The design of a broadband 4-mm magic-angle spinning (MAS) X-(1)H/(19)F double resonance probe for cross-polarization (CP)/MAS NMR studies at 21.15 T ((1)H at 900 MHz) is described. The high-frequency (1)H/(19)F channel employs a new and efficient transmission line tuning design. The first (13)C CP/MAS NMR spectra recorded at 21.15 T have been obtained with this probe and exhibit the best S/N per milligram sample of hexamethylbenzene achieved so far for a 4-mm rotor.     

Sensitivity enhancement, assignment, and distance measurement in 13C solid-state NMR spectroscopy for paramagnetic systems under fast magic angle spinning

Despite success of previous studies, high-resolution solid-state NMR (SSNMR) of paramagnetic systems has been still largely unexplored because of limited sensitivity/resolution and difficulty in assignment due to large paramagnetic shifts. Recently, we demonstrated that an approach using very-fast magic angle spinning (VFMAS; spinning speed 20kHz) enhances resolution/sensitivity in (13)C SSNMR for paramagnetic complexes [Y. Ishii, S. Chimon, N.P. Wickramasinghe, A new approach in 1D and 2D (13)C high resolution solid-state NMR spectroscopy of paramagnetic organometallic complexes by very fast magic-angle spinning, J. Am. Chem. Soc. 125 (2003) 3438-3439]. In this study, we present a new strategy for sensitivity enhancement, signal assignment, and distance measurement in (13)C SSNMR under VFMAS for unlabeled paramagnetic complexes using recoupling-based polarization transfer. As a robust alternative of cross-polarization (CP), rapid application of recoupling-based polarization transfer under VFMAS is proposed. In the present approach, a dipolar-based analog of INEPT (dipolar INEPT) methods is used for polarization transfer and a (13)C signal is observed under VFMAS without (1)H decoupling. The resulting low duty factor permits rapid signal accumulation without probe arcing at recycle times ( approximately 3 ms/scan) matched to short (1)H T(1) values of small paramagnetic systems ( approximately 1 ms). Experiments on Cu(dl-Ala)(2) showed that the fast repetition approach under VFMAS provided sensitivity enhancement by a factor of 8-66 for a given sample, compared with the (13)C MAS spectrum under moderate MAS at 5kHz. The applicability of this approach was also demonstrated for a more challenging system, Mn(acac)(3), for which (13)C and (1)H paramagnetic shift dispersions reach 1500 and 700 ppm, respectively. It was shown that effective-evolution-time dependence of transferred signals in dipolar INEPT permitted one to distinguish (13)CH, (13)CH(2), (13)CH(3), (13)CO2- groups in 1D experiments for Cu(DL-Ala)(2) and Cu(Gly)(2). Applications of this technique to 2D (13)C/(1)H correlation NMR under VFMAS yielded reliable assignments of (1)H resonances as well as (13)C resonances for Cu(DL-Ala)(2) and Mn(acac)(3). Quantitative analysis of cross-peak intensities in 2D (13)C/(1)H correlation NMR spectra of Cu(DL-Ala)(2) provided distance information between non-bonded (13)C-(1)H pairs in the paramagnetic system.     

Sensitivity enhancement of NMR spectra of half-integer spin quadrupolar nuclei in solids using hyperbolic secant pulses

The experimental factors influencing the enhancements achievable for the central NMR transition, m(I)=1/2-->m(I)=-1/2, of spin-3/2 and spin-5/2 nuclei in the solid state using hyperbolic secant, HS, pulses for population transfer are investigated. In the case of powder samples spinning at the magic angle, it is found that the spinning frequency, the bandwidth and the frequency offset of the HS pulse play a crucial role in determining the maximum enhancements. Specifically, the bandwidth must be set to the spinning frequency for maximum signal enhancements. The (87)Rb NMR enhancement obtained for RbClO(4) using HS pulses was relatively insensitive to the magic angle spinning frequency; however, in the case of Al(acac)(3), the (27)Al enhancement increased with MAS frequency. In order to obtain an adiabatic HS sweep, one should optimize the rf field for a given pulse duration or optimize the pulse duration for a given rf field.     

2CALIS doubling the sensitivity of CALIS for calibration of the rf field strength for indirectly observed nuclei

A new set of pulse sequences, 2CALIS, that exhibit double sensitivity of the recent CALIS pulse sequences for accurate calibration of the rf field strength for an indirectly observed spin is introduced. The sensitivity gain is a result of not forming heteronuclear coherence transfer gradient echoes although they are excellent for artifact suppression. It is, however, demonstrated that the scheme in 2CALIS for suppression of non (13)C-attached proton magnetization is adequate for calibration of the (13)C rf field strength even on natural abundance samples. A 2CALIS version with Watergate applicable to biomolecules in aqueous solution is also presented and demonstrated both in (13)C natural abundance and on a (13)C, (15)N enriched protein sample.     

Methylene spectral editing in solid-state 13C NMR by three-spin coherence selection

A robust new solid-state nuclear magnetic resonance (NMR) method for selecting CH2 signals in magic-angle spinning (MAS) 13C NMR spectra is presented. Heteronuclear dipolar evolution for a duration of 0.043 ms, under MREV-8 homonuclear proton decoupling, converts 13C magnetization of CH2 groups into two- and three-spin coherences. The CH2 selection in the SIJ (C H H) spin system is based on the three-spin coherence S(x)I(z)J(z), which is distinguished from 13C magnetization (S(x)) by a 1H 0 degrees/90 degrees pulse consisting of two 45 degrees pulses. The two-spin coherences of the type S(y)I(z) are removed by a 13C 90 degrees x-pulse. The three-spin coherence is reconverted into magnetization during the remainder of the rotation period, still under MREV-8 decoupling. The required elimination of 13C chemical-shift precession is achieved by a prefocusing 180 degrees pulse bracketed by two rotation periods. The selection of the desired three-spin coherence has an efficiency of 13% theoretically and of 8% experimentally relative to the standard CP/MAS spectrum. However, long-range couplings also produce some three-spin coherences of methine (CH) carbons. Therefore, the length of the 13C pulse flipping the two-spin coherences is increased by 12% to slightly invert the CH signals arising from two-spin coherences and thus cancel the signal from long-range three-spin coherences. The signal intensity in this cleaner spectrum is 6% relative to the regular CP/TOSS spectrum. The only residual signal is from methyl groups, which are suppressed at least sixfold relative to the CH2 peaks. The experiment is demonstrated on cholesteryl acetate and applied to two humic acids.     

HR/MAS高分辨魔角微量探头用于天然橡胶固体1H、13C NMR测试及其与液体BBO探头、固体CP/MAS探头测试的比较

采用BRUKER高分辨魔角微量探头（HR/MAS），液相宽带BBO探头和固体CP/MAS探头，对天然橡胶固体、乳液以及天然橡胶溶于氘代苯的溶液进行了¹H、¹³C 1D和2D NMR谱的测试和比较. 发现HR/MAS探头用于天然橡胶固体和乳液时可以得到高分辨的¹H、¹³C谱，克服了CP/MAS探头测试固体¹³C NMR谱或者是固体¹H NMR谱时，谱图存在S/N值可能较小、谱峰可能宽化的弱点.     

A 2D MAS solid-state NMR method to recover the amplified heteronuclear dipolar and chemical shift anisotropic interactions

A two-dimensional solid-state NMR method for the measurement of chemical shift anisotropy tensors of X nuclei (15N or 13C) from multiple sites of a polypeptide powder sample is presented. This method employs rotor-synchronized pi pulses to amplify the magnitude of the inhomogeneous X-CSA and 1H-X dipolar coupling interactions. A combination of on-resonance and magic angle rf irradiation of protons is used to vary the ratio of the magnitudes of the 1H-X dipolar and X-CSA interactions which are recovered under MAS, in addition to suppressing the 1H-1H dipolar interactions. The increased number of spinning sidebands in the recovered anisotropic interactions is useful to determine the CSA tensors accurately. The performance of this method is examined for powder samples of N-acetyl-(15)N-L-valine (NAV), N-acetyl-15N-L-valyl-15N-L-leucine (NAVL), and alpha-13C-L-leucine. The sources of experimental errors in the measurement of CSA tensors and the application of the pulse sequences under high-field fast MAS operations are discussed.     

Investigations of low-amplitude radio frequency pulses at and away from rotary resonance conditions for I = 5/2 nuclei

Additional experimental evidence of rotary resonance effects for multiple-quantum coherence conversion in a spin-5/2 system is presented. Two-dimensional plots of the relative efficiency of MQ excitation and conversion are given as a function of radio frequency (rf) amplitude and pulse width. Data are presented for the excitation of five-quantum coherence (5QC), as well as for 5QC to three-quantum coherence (3QC) conversion, 5QC to IQC (the central transition coherence) conversion, and 3QC to IQC conversion. A two-fold increase in the signal-to-noise ratio is achieved by substituting low amplitude rf pulses in place of hard rf pulses for 5QC excitation and 5QC to 3QC conversion in a mixed multiple-quantum magic angle spinning (MAS) (MMQMAS) experiment. The anisotropic line shape for the low-amplitude rf pulse version of the MMQMAS experiment was observed to be distorted from the MAS line shape. The cause and implications of the distortion are discussed.     

High resolution 2D 1H-13C correlation of cholesterol in model membrane

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.     

Heteronuclear decoupling under fast MAS by a rotor-synchronized Hahn-echo pulse train

A new heteronuclear decoupling mechanism under fast magic-angle spinning MAS is introduced. It is based on refocusing the coherences responsible for the dephase of low-gamma nuclei ((13)C, (15)N) transverse spin-polarization in the presence of strongly dipolar-coupled protons, and has the advantage that can be implemented by pulsed techniques, with all the benefits resulting from a reduced duty cycle compared with conventional decoupling by continuous rf irradiation. The decoupling efficiency of a simple rotor-synchronized Hahn-echo pulse train is analyzed both theoretically and experimentally. It was found that a substantial improvement in sensitivity and resolution can be achieved in compounds with small (1)H chemical shielding parameters even at moderate sample spinning, and some interesting applications are shown. It is also shown that much faster spinning frequencies, or alternative refocusing sequences, are needed for applications on rigid organic solids, i.e., in systems with larger (1)H chemical shifts.     

Sensitivity considerations in polarization transfer and filtering using dipole-dipole couplings: implications for biomineral systems

The robustness and sensitivities of different polarization-transfer methods that exploit heteronuclear dipole-dipole couplings are compared for a series of heterogeneous solid systems, including polycrystalline tetrakis(trimethylsilyl)silane (TKS), adamantane, a physical mixture of doubly (13)C,(15)N-enriched and singly (13)C-enriched polycrystalline glycine, and a powder sample of siliceous marine diatoms, Thalossiosira pseudonana. The methods were analyzed according to their respective frequency-matching spectra or resultant signal intensities. For a series of (13)C{(1)H} cross-polarization experiments, adiabatic passage Hartmann-Hahn cross-polarization (APHH-CP) was shown to have several advantages over other methods, including Hartmann-Hahn cross-polarization (HHCP), variable-amplitude cross-polarization (VACP), and ramped-amplitude cross-polarization (RACP). For X-Y systems, such as (13)C{(15)N}, high and comparable sensitivities were obtained by using APHH-CP with Lee-Goldburg decoupling or by using the transferred-echo double resonance (TEDOR) experiment. The findings were applied to multinuclear (1)H, (13)C, (15)N, and (29)Si CP MAS characterization of a powder diatom sample, a challenging inorganic-organic hybrid solid that places high demands on NMR signal sensitivity.     

A solid-state NMR application of the anomeric effect in carbohydrates: galactosamine, glucosamine, and N-acetyl-glucosamine

Simple 2D 13C/15N heteronuclear correlation solid-state NMR spectroscopy was implemented to resolve the 15N resonances of the alpha and beta anomers of three amino monosaccharides: galactosamine (GalN), glucosamine hydrochloride (GlcN), and N-acetyl-glucosamine (GlcNAc) labeled specifically with 13C1/15N spin pairs. Although the 15N resonances could not be distinguished in normal 1D spectra, they were well resolved in 2D double CP/MAS correlation spectra by taking advantage of the 13C spectral resolution. The alpha and beta resonances shifted apart by 3-5 ppm in their 13C chemical shifts, and differed by 1-2 ppm in the extended 15N dimension. Aside from this, the detection of other 13C/15N correlations over short distances was also achieved arising from the C2, C3 and CO carbons present in natural abundance. 2D double CP/MAS chemical shift correlation NMR spectroscopy is a simple and powerful technique to characterize the anomeric effect of amino monosaccharides. Applications of the 2D method reveal well-resolved 15N and 13C chemical shifts might be useful for structural determination on carbohydrates of biological significance, such as glycopeptide or glycolipids.     

Rapid solid-state NMR of deuterated proteins by interleaved cross-polarization from ¹H and ²H nuclei

We present a novel sampling strategy, interleaving acquisition of multiple NMR spectra by exploiting initial polarization subsequently from (1)H and (2)H spins, taking advantage of their different T(1) relaxation times. Different (1)H- and (2)H-polarization based spectra are in this way simultaneously recorded improving either information content or sensitivity by adding spectra. The so-called Relaxation-optimized Acquisition of Proton Interleaved with Deuterium (RAPID) (1)H→(13)C/(2)H→(13)C CP/MAS multiple-acquisition method is demonstrated by 1D and 2D experiments using a uniformly (2)H, (15)N,(13)C-labeled α-spectrin SH3 domain sample with all or 30% back-exchanged labile (2)H to (1)H. It is demonstrated how 1D (13)C CP/MAS or 2D (13)C-(13)C correlation spectra initialized with polarization from either (1)H or (2)H may be recorded simultaneously with flexibility to be added or used individually for spectral editing. It is also shown how 2D (13)C-(13)C correlation spectra may be recorded interleaved with (2)H-(13)C correlation spectra to obtain (13)C-(13)C correlations along with information about dynamics from (2)H sideband patterns.     

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.     

Dynamic nuclear polarization at 9T using a novel 250GHz gyrotron microwave source

In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9T (250 GHz for g=2 electrons, 380 MHz for 1H). In these experiments, 1H enhancements of up to 170+/-50 have been observed in 1-13C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20K; in addition, we have observed significant enhancements in 15N spectra of unoriented pf1-bacteriophage. Finally, enhancements of approximately 17 have been obtained in two-dimensional 13C-13C chemical shift correlation spectra of the amino acid U-13C, 15N-proline during magic angle spinning (MAS), demonstrating the stability of the DNP experiment for sustained acquisition and for quantitative experiments incorporating dipolar recoupling. In all cases, we have exploited the thermal mixing DNP mechanism with the nitroxide radical 4-amino-TEMPO as the paramagnetic dopant. These are the highest frequency DNP experiments performed to date and indicate that significant signal enhancements can be realized using the thermal mixing mechanism even at elevated magnetic fields. In large measure, this is due to the high microwave power output of the 250 GHz gyrotron oscillator used in these experiments.     

F/Si distance determination in fluoride-containing octadecasil by Hartmann–Hahn cross-polarization under fast magic-angle spinning

¹⁹F/²⁹Si Hartmann–Hahn continuous wave cross-polarization (CP) has been applied under fast magic-angle spinning (MAS) to a powder sample of octadecasil. Strong oscillations occur during CP on a sideband matching condition between the isolated ²⁹Si–¹⁹F spin pairs formed by the silicons in the D4R units and the fluoride anions. The magnitude of the dipolar coupling constant was deduced directly from the line-splitting between the intense singularities of the Pake-like patterns obtained by Fourier transformation of the oscillatory polarization transfer. The corresponding Si–F internuclear distance, r=2.62±0.05 Å, is found to be in very good agreement with the X-ray crystal structure and the value of 2.69±0.04 Å recently reported from rotational echo double resonance (REDOR) and transferred echo double resonance (TEDOR) nuclear magnetic resonance (NMR) experiments. Furthermore, the CP technique is still reliable under fast MAS where both REDOR and TEDOR sequences suffer from severe artefacts due to finite pulse lengths. In octadecasil, a spinning frequency of 14 kHz is shown to be necessary for an effective suppression of ¹⁹F–¹⁹F spin diffusion. The influences of experimental missettings and radiofrequency (RF) field inhomogeneity are taken into account.     

Coherence transfer between spy nuclei and nitrogen-14 in solids

Coherence transfer from 'spy nuclei' such as (1)H or (13)C (S=1/2) was used to excite single- or double-quantum coherences of (14)N nuclei (I=1) while the S spins were aligned along the static field, in the manner of heteronuclear single-quantum correlation (HSQC) spectroscopy. For samples spinning at the magic angle, coherence transfer can be achieved through a combination of scalar couplings J(I,S) and second-order quadrupole-dipole cross-terms, also known as residual dipolar splittings (RDS). The second-order quadrupolar powder patterns in the two-dimensional spectra allow one to determine the quadrupolar parameters of (14)N in amino acids.     

13C high-resolution solid state NMR studies of the structure and dynamics of 1,6:3,4-dianhydro-2-O-tosyl-beta-D-galactopyranose

13C CP/MAS, dipolar dephasing MAS and theoretical GIAO calculations were employed to assign 13C resonances to the molecular structure of 1,6:3,4-dianhydro-2-O-tosyl-beta-D-galactopyranose 1. From spinning sideband intensities, employing the graphical method of Herzfeld and Berger the 13C delta(ii) parameters for aromatic residue were calculated. The experimental data were compared with computed results obtained by means of the B3PW91 hybrid method and 6-311G (df, p) basis set. The X-ray geometry of 1 with the correlated position of hydrogen atoms was taken as input data for theoretical calculations. As concluded from Cambridge Crystallographic Database (CSD) search, there are two reports describing the X-ray studies of 1 that show the slightly different geometry of the compound under investigation. This work shows that such discrepancies in geometry can generate differences between computed 13C delta(ii) parameters up to 6 ppm. 13C T1 and 1H T1rho relaxation times reveal that 1 is very rigid in crystal lattice. This structure is characterized by extremely long 1H T1rho, found to be in range ca. 200 ms.     

Characterization of 15N chemical shift tensors via 15N-13C REDOR and 1N-1H dipolar-shift CPMAS NMR spectroscopy

As part of our studies on the characterization of 15N chemical shift anisotropy (CSA) via magic angle spinning (MAS) NMR spectroscopy, we have investigated via numerical simulations the sensitivity of two different REDOR experimental protocols to the angles defining the orientation of the 15N-13C' bond vector in the principal axis system of the 15N CSA tensor of the amide nitrogen in a peptide bond. Additionally, employing polycrystalline samples of 15N and 13C', 15N-labeled acetanilide, we have obtained, in a first study of this type, the orientation of the 15N CSA tensor in the molecular frame by orienting the tensor with respect to the 15N-3C' and 15N-1H dipolar vectors via 15N-13C' REDOR and 15N-1H dipolar-shift MAS experiments, respectively.