Design and construction of a contactless mobile RF coil for double resonance variable angle spinning NMR

Variable angle spinning (VAS) experiments can be used to measure long-range dipolar couplings and provide structural information about molecules in oriented media. We present a probe design for this type of experiment using a contactless resonator. In this circuit, RF power is transmitted wirelessly via coaxial capacitive coupling where the coupling efficiency is improved by replacing the ordinary sample coil with a double frequency resonator. Our probe constructed out of this design principle has shown favorable properties at variable angle conditions. Moreover, a switched angle spinning correlation experiment is performed to demonstrate the probe's capability to resolve dipolar couplings in strongly aligned molecules.     

Variable angle NMR spectroscopy and its application to the measurement of residual chemical shift anisotropy

The successful measurement of anisotropic NMR parameters like residual dipolar couplings (RDCs), residual quadrupolar couplings (RQCs), or residual chemical shift anisotropy (RCSA) involves the partial alignment of solute molecules in an alignment medium. To avoid any influence of the change of environment from the isotropic to the anisotropic sample, the measurement of both datasets with a single sample is highly desirable. Here, we introduce the scaling of alignment for mechanically stretched polymer gels by varying the angle of the director of alignment relative to the static magnetic field, which we call variable angle NMR spectroscopy (VA-NMR). The technique is closely related to variable angle sample spinning NMR spectroscopy (VASS-NMR) of liquid crystalline samples, but due to the mechanical fixation of the director of alignment no sample spinning is necessary. Also, in contrast to VASS-NMR, VA-NMR works for the full range of sample inclinations between 0° and 90°. Isotropic spectra are obtained at the magic angle. As a demonstration of the approach we measure 13C-RCSA values for strychnine in a stretched PDMS/CDCl? gel and show their usefulness for assignment purposes. In this context special care has been taken with respect to the exact calibration of chemical shift data, for which three approaches have been derived and tested.     

Recoupling of residual dipolar couplings in single-domain polymer-stabilized liquid crystals undergoing magic-angle spinning

Measurement of dipolar couplings, chemical shift anisotropies, and quadrupole couplings in oriented media such as liquid crystals are of great importance for extraction of structural parameters in biological macromolecules. Here, we introduce a new technique, SAD-REDOR, that consists of recoupling heteronuclear dipolar couplings in molecules dissolved in a single-domain liquid crystal or other oriented medium through the combined use of magic-angle spinning and rotor-synchronized radiofrequency pulses. This application of the REDOR pulse sequence to oriented media offers several advantages such as selectivity over the type of coupling recovered and tunable scaling of the interaction. The effectiveness of the technique is demonstrated both theoretically and experimentally, using the recently developed polyacrylamide-stabilized Pf1 phage medium and 15N-labeled benzamide as the aligned molecule.     

Multiple quantum nuclear magnetic resonance spectra of partially oriented styrene in a nematic phase

Proton multiple-quantum nuclear magnetic resonance (MQNMR) spectra of styrene partially oriented in a nematic liquid crystal were acquired using the magnetic field gradient method. Six-, seven- and eight-quantum spectra were iteratively solved to yield all intramolecular dipolar couplings (D_ij) which were used to determine the molecular shape of styrene. The dipolar couplings were fitted to a model averaging between four equivalent non-planar conformations of the molecule. Ignoring vibrational corrections, the analysis was consistent with a non-planar molecular geometry with a dihedral angle of approximately 16 degrees between the olefinic fragment and the aromatic ring.     

General Features of the X Part of an ABX Spin System in Isotropic and Liquid Crystalline Phases as Illustrated by theC–{H} Spectra of 2,2′-Difluorobiphenyl

The¹³C–{¹H} NMR spectra of 2,2′-difluorobiphenyl dissolved in isotropic and liquid crystalline solvents have been obtained and analyzed. They are examples of the X part of an ABX spectrum. It is shown that the spectrum of the isotropic solution yieldsJ_AX,J_BX,J_AB, δ_AB, and δ_X, but only if all the transitions are detected, and that intensities as well as frequencies of the transitions are used in the analysis. It is demonstrated that for 2,2′-difluorobiphenyl this requires that for some of the carbons it is necessary to detect very weak transitions. For the spectra of liquid crystalline solutions of ABX systems it is shown that the dipolar couplingsD_AX,D_BX, andD_ABare obtained only if these couplings are in a certain sensitive range of relative values. The sensitive range can be adjusted by using variable angle sample spinning (VASS). It is demonstrated that VASS spectra taken near the magic angle can be used to obtain the absolute signs of the scalar couplings.     

Distribution effects on 1H double-quantum MAS NMR spectra

The effect of a distribution in the (1)H-(1)H dipolar coupling on (1)H double-quantum (DQ) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spinning sideband patterns is considered. In disordered or amorphous materials a distribution in the magnitude of the (1)H-(1)H dipolar coupling is a realistic possibility. Simulations of the (1)H DQ MAS NMR spinning sideband spectra were performed with the two-spin approximation. These simulations reveal that a dipolar coupling distribution can greatly affect the DQ spectral shape and behavior of the DQ build-up. The spectral line shapes are quantified by measurement of the relative intensities of the DQ sidebands. These variations in the (1)H DQ NMR spectra are evaluated as a function of the width of the dipolar coupling distribution. As an example, the experimental DQ spinning sideband spectrum for a hydrated polyoxoniobate containing 15 H(2)O molecules per hexaniobate cluster, are better simulated with a distribution of dipolar couplings opposed to a single coupling constant.     

1H–13C hetero-nuclear dipole–dipole couplings of methyl groups in stationary and magic angle spinning solid-state NMR experiments of peptides and proteins

¹³C NMR of isotopically labeled methyl groups has the potential to combine spectroscopic simplicity with ease of labeling for protein NMR studies. However, in most high resolution separated local field experiments, such as polarization inversion spin exchange at the magic angle (PISEMA), that are used to measure ¹H–¹³C hetero-nuclear dipolar couplings, the four-spin system of the methyl group presents complications. In this study, the properties of the ¹H–¹³C hetero-nuclear dipolar interactions of ¹³C-labeled methyl groups are revealed through solid-state NMR experiments on a range of samples, including single crystals, stationary powders, and magic angle spinning of powders, of ¹³C₃ labeled alanine alone and incorporated into a protein. The spectral simplifications resulting from proton detected local field (PDLF) experiments are shown to enhance resolution and simplify the interpretation of results on single crystals, magnetically aligned samples, and powders. The complementarity of stationary sample and magic angle spinning (MAS) measurements of dipolar couplings is demonstrated by applying polarization inversion spin exchange at the magic angle and magic angle spinning (PISEMAMAS) to unoriented samples.     

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].     

Magnetic field gradients in solid state magic angle spinning NMR.

Magnetic field gradients have proven useful in NMR for coherence pathway selection, diffusion studies, and imaging. Recently they have been combined with magic angle spinning to permit high-resolution measurements of semi-solids, where magic angle spinning averages any residual dipolar couplings and local variations in the bulk magnetic susceptibility. Here we show the first examples of coherence pathway selection by gradients in dipolar coupled solids. When the gradient evolution competes with dipolar evolution the experiment design must take into account both the strength of the dipolar couplings and the means to refocus it. Examples of both homonuclear and heteronuclear experiments are shown in which gradients have been used to eliminate the need for phase cycling.     

Experimental aspects of multidimensional solid-state NMR correlation spectroscopy.

The experimental parameters critical for the implementation of multidimensional solid-state NMR experiments that incorporate heteronuclear spin exchange at the magic angle are discussed. This family of experiments is exemplified by the three-dimensional experiment that correlates the (1)H chemical shift, (1)H-(15)N dipolar coupling, and (15)N chemical shift frequencies. The broadening effects of the homonuclear (1)H-(1)H dipolar couplings are suppressed using flip-flop (phase- and frequency-switched) Lee-Goldburg irradiations in both the (1)H chemical shift and the (1)H-(15)N dipolar coupling dimensions. The experiments are illustrated using the (1)H and (15)N chemical shift and dipolar couplings in a single crystal of (15)N-acetylleucine.     

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.     

Ordering in nematic liquid crystals from NMR cross-polarization studies

The measurement of dipolar couplings between nuclei is a convenient way of obtatining directly liquid crystalline ordering through NMR since the coupling is dependent on the average orientation of the dipolar vector in the magnetic field which also aligns the liquid crystal. However, measurement of the dipolar coupling between a pair of selected nuclei is beset with problems that require special solutions. In this article the use of cross polarization for measuring dipolar couplings in liquid crystals is illustrated. Transient oscillations observed during cross polarization provide the dipolar couplings between essentially isolated nearest neighbour spins which can be extracted for several sites simultaneously by employing two-dimensional NMR techniques. The use of the method for obtaining heteronuclear dipolar couplings and hence the order parameters of liquid crystals is presented. Several modifications to the basic experiment are considered and their utility illustrated. A method for obtaining proton-proton dipolar couplings, by utilizing cross polarization from the dipolar reservoir, is also presented.     

Capture and manipulation of magnetically aligned Pf1 with an aqueous polymer gel

The magnetic alignment of the Pseudomonas bacteriophage Pf1 is captured indefinitely in a gel of the aqueous triblock copolymer Pluronic F-127. In addition to preserving high-resolution liquids NMR spectra for dissolved solutes, the gel prevents the reorientation of the phage allowing mechanical manipulation of the angle between the axis of the phage alignment and the static magnetic field. The residual 2H quadrupolar couplings for several solutes dissolved in this material as a function of the angle Theta between the non-spinning sample tube and the static magnetic field are consistent with the value of P(2)(cosTheta)=(3cos(2)Theta-1)/2. The variable-angle correlation spectrum for these solutes is shown to separate residual quadrupolar effects from isotropic chemical shifts. Finally, the compatibility of Pluronic F-127 with NMR studies of aqueous biological macromolecules is demonstrated in a measurement of residual dipolar couplings in an 15N-labeled nucleic acid.     

Two Simple NMR Experiments for Measuring Dipolar Couplings in Asparagine and Glutamine Side Chains

Residual dipolar couplings are now widely used for structure determination of biological macromolecules. Until recently, the main focus has been on measurement of dipolar couplings in the protein main chain. However, with the aim of more complete protein structure, it is also essential to have information on the orientation of protein side chains. In addition, residual dipolar couplings can potentially be employed to study molecular dynamics. In this Communication, two simple NH(2) and spin-state edited experiments are presented for rapid and convenient determination of five residual dipolar couplings from (15)N, (1)H correlation spectrum in asparagine and glutamine side chains. The pulse sequences are demonstrated on two proteins, 30.4-kDa Cel6A in diluted liquid crystal phase and 18-kDa human cardiac troponin C in water.     

Solid state NMR analysis of the dipolar couplings within and between distant CF3-groups in a membrane-bound peptide

Dipolar couplings contain information on internuclear distances as well as orientational constraints. To characterize the structure of the antimicrobial peptide gramicidin S when bound to model membranes, two rigid 4-CF3-phenylglycine labels were attached to the cyclic backbone such that they reflect the behavior of the entire peptide. By solid state 19F NMR we measured the homonuclear dipolar couplings of the two trifluoromethyl-groups in oriented membrane samples. Using the CPMG experiment, both the strong couplings within each CF3-group as well as the weak coupling between the two CF3-groups could be detected. An intra-CF3-group dipolar coupling of 86 Hz and a weak inter-group coupling of 20 Hz were obtained by lineshape simulation of the complex dipolar spectrum. It is thus possible to explore the large distance range provided by 19F-labels and to resolve weak dipolar couplings even in the presence of strong intra-CF3 couplings. We applied this approach to distinguish and assign two epimers of the labeled gramicidin S peptide on the basis of their distinct 19F dipolar coupling patterns.     

Analyses of the complex proton NMR spectra: determination of anisotropic proton chemical shifts of oriented molecules by a two dimensional experiment

NMR spectra of molecules oriented in the liquid crystalline media provide information on the molecular structure and order parameter. However, the numerical iterative analysis of the proton spectra of strongly coupled spins is difficult and time consuming. Such analysis is simplified if nearly accurate starting parameters are available. One such parameter is the chemical shift which in the oriented media is very different from the isotropic values due to anisotropic contributions. In this study, we have explored the possibility of obtaining chemical shifts in the oriented phase to aid the analysis of the spectra. A two dimensional experiment in which FSLG decoupling employed during the t1 period eliminates the homonuclear dipolar couplings and retains only the chemical shifts has been implemented. Experiments on the molecule cis,cis-mucononitrile demonstrate that the chemical shifts obtained by this procedure are nearly the same as the chemical shifts derived by iterative analysis of the one dimensional spectrum of the molecule following the standard procedure. The method has also been used to analyse the spectrum of 1-iodopropane using the chemical shifts obtained from the proposed experiment as the starting parameters.     

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.     

Homonuclear correlation experiments for quadrupolar nuclei, spinning away from the magic angle

We present a set of homonuclear correlation experiments for half-integer quadrupolar spins in solids. In all these exchange-type experiments, the dipolar interaction is retained during the mixing time by spinning the sample at angles other than the “regular magic angle” (54.7°). The second-order quadrupolar interaction is averaged by different strategies for the different experiments. The multiple-quantum off magic angle spinning (MQOMAS) exchange experiment is essentially a regular MQMAS experiment where the quadrupolar interaction is averaged by combining magic angle spinning with a multiple- to single-quantum correlation scheme. The sample is spun at the magic angle at all times except during the mixing time which is added to establish homonuclear correlation. In the multiple-quantum P₄ magic angle spinning (MQP₄MAS) exchange experiment, the sample is spun at one of the angles at which the fourth-order Legendre polynomial vanishes (P₄ magic angle), the remaining second-order quadrupolar interaction now governed by a second-rank tensor is refocussed by the multiple to single-quantum correlation scheme. In the dynamic angle spinning (DAS) exchange experiment, the second-order quadrupolar interaction is averaged by correlating the evolution from two complementary angles. These experiments are demonstrated and compared, in view of their specific advantages and disadvantages, for ²³Na in the model compound Na₂SO₃.     

Through-bond connectivity in solids by continuous-wave spin lock

A simple two-dimensional correlation experiment that enables determination of through-bond connectivity in the solid state is described. The experiment is performed under fast magic angle spinning (MAS) conditions. After the initial pi/2 pulse, the magnetization develops freely under the MAS Hamiltonian. The t1-period is followed by a strong spin locking pulse used as mixing period. The dipolar coupling is averaged out by magic angle spinning, and the chemical shifts and r.f.-offsets are scaled by the applied spin locking field. Hence, for strong locking conditions, the isotropic J-coupling is the dominant interaction. The mixing Hamiltonian is thus identical to the well-known TOCSY-Hamiltonian, resulting in a net through-bond magnetization transfer. The mixing-time dependence of the exchange rates is investigated. Applications to crystalline P4S7 and MgP4O11 are shown.     

Broadband Polarization Transfer under Magic-Angle Spinning: Application to Total Through-Space-Correlation NMR Spectroscopy

A pulse sequence is described that leads to a broadband recoupling of the dipolar interaction in magic-angle-spinning solid-state NMR experiments of¹³C spins. The sequence is based on a combination of rotating frame and laboratory frame transfer periods. The recovered dipolar interaction is only weakly dependent on spectral parameters but is a faithful measure for the internuclear distances. Furthermore, a pure zero-quantum term is recovered (of the type found in static “spin-diffusion” experiments). This makes the pulse sequence particularly suited for incorporation into two-dimensional total through-space correlation experiments that deliver simultaneous information about all dipolar couplings in a single 2D experiment. It is found that the necessary decoupling from abundant protons is best performed in two steps: first, the strong homonuclear couplings between the high-γ spins are averaged by Lee–Goldburg irradiation and, second, the heteronuclear dipolar interaction is averaged by the combined application of an RF field to the low-γ spins and magic-angle sample spinning. Phase-inversion and amplitude attenuation in the rotating frame and refocusing pulses in the laboratory frame part of the pulse sequence are introduced to achieve an optimum chemical-shift offset-independence and for the suppression of unwanted double-quantum transitions. The design principles are explained in detail. Finally, the pulse scheme is applied to total-correlation spectroscopy of a uniformly labeled amino acid. The experimental cross-peak intensities are in qualitative agreement with the known crystal structure of the model compound.