Investigation of the dipolar dephasing NMR on organic solids

We present a method based on integro-differential equations describing the¹³C?¹H dipolar dephasing behaviour of carbon magnetization which results from monoprotonated carbons, non-protonated carbons as well as rapidly rotating methyl groups. Good agreement with theoretical calculations and experiment is obtained in ammonium tartrate and durene. The frequently applied empirical methods for determination the ratio of protonated and non-protonated carbons are analyzed. The dipolar dephasing time constants of non-protonated carbons vary substantially as a result, of variation in their heteronuclear second moments and thus in structure. Two different methods are performed for determination heteronuclear second moments from dipolar dephasing data.     

A novel dipolar dephasing method for the slow magic angle turning experiment

Complete suppression of the resonances from protonated carbons in a slow magic angle spinning experiment can be achieved using five dipolar dephasing (Five-DD) periods distributed in one rotor period. This produces a spectrum containing only the spinning sidebands (SSB) from the nonprotonated carbons. It is shown that the SSB patterns corresponding to the nonprotonated carbons are not distorted over a wide range of dipolar dephasing times. Hence, this method can be used to obtain reliable principal values of the chemical shift tensors for each nonprotonated carbon. The Five-DD method can be readily incorporated into isotropic-anisotropic 2D experiments such as FIREMAT and 2D-PASS to facilitate the measurement of the (13)C chemical shift tensors in complex systems.     

Recoupled long-range C-H dipolar dephasing in solid-state NMR,and its use for spectral selection of fused aromatic rings

This work introduces a simple new solid-state 13C NMR method for distinguishing various types of aromatic residues, e.g. those of lignin from fused rings of charcoal. It is based on long-range dipolar dephasing, which is achieved by recoupling of long-range C-H dipolar interactions, using two 1H 180 degrees pulses per rotation period. This speeds up dephasing of unprotonated carbon signals approximately threefold compared to standard dipolar dephasing without recoupling and thus provides much more efficient differential dephasing. It also reduces the effects of spinning-speed dependent effective proton-proton dipolar couplings on the heteronuclear dephasing. Signals of unprotonated carbons with two or more protons at a two-bond distance dephase to <3% within less than 0.9 ms, significantly faster than those of aromatic sites separated from the nearest proton by three or more bonds. Differential dephasing among different unprotonated carbons is demonstrated in a substituted anthraquinone and 3-methoxy benzamide. The data yield a calibration curve for converting the dephasing rates into estimated distances from the carbon to the nearest protons. This can be used for peak assignment in heavily substituted or fused aromatic molecules. Compared to lignin, slow dephasing is observed for the aromatic carbons in wood charcoal, and even slower for inorganic carbonate. Direct 13C polarization is used on these structurally complex samples to prevent loss of the signals of interest, which by design originate from carbons that are distant from protons and therefore crosspolarize poorly. In natural organic matter such as humic acids, this combination of recoupled dipolar dephasing and direct polarization at 7-kHz MAS enables selective observation of signals from fused rings that are characteristic of charcoal.     

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].     

Quantitative characterization of coal structure by high-resolution CP/MAS 13C solid-state NMR spectroscopy

A method for quantitatively characterizing the carbon skeletal structure of coal by variable contact time experiment using high-resolution CP/MAS ¹³C solid-state NMR spectroscopy is proposed in this paper. The initial polarization transfer intensity from protons directly bonded with carbons, instead of dipolar-dephasing techniques which had to run on a lower frequency NMR spectrometer (100.02 MHz for proton), was used to divide the bridgehead and protonated aromatic carbons, making all the NMR data in this paper obtained on a high frequency NMR spectrometer (500.12 MHz for proton). On this basis, the fractions of different carbons in coal were further divided by the initial polarization transfer intensity from spin diffusion of protons unbonded with carbons. The structure of Naomaohu coal, a subbituminous coal from China, was measured. The change of polarization transfer with contact time was analyzed quantitatively. The fractions of aromatic, aliphatic, carboxyl and carbonyl carbons, and corrective aromaticity are 0.61, 0.39, 0.1 and 0.51, respectively. The fractions of protonated and bridgehead aromatic carbons are 0.22 and 0.09, respectively. These results agreed with literatures, and bond concentration calculated by the carbon skeletal structure distribution of coal was reasonable.     

Separation of aromatic-carbon 13C NMR signals from di-oxygenated alkyl bands by a chemical-shift-anisotropy filter

Selection of alkyl-carbon and suppression of aromatic-carbon 13C nuclear magnetic resonance (NMR) signals has been achieved by exploiting the symmetry-based, systematic difference in their 13C chemical-shift anisotropies (CSAs). Simple three- or five-pulse CSA-recoupling sequences with "gamma-integral" cleanly suppress the signals of all sp2- and sp-hybridized carbons. The chemical-shift-anisotropy-based dephasing is particularly useful for distinguishing the signals of di-oxygenated alkyl (O-C-O) carbons, found for instance as anomeric carbons in carbohydrates, from bands of aromatic carbons with similar 13C isotropic chemical shifts. The alkyl signals are detected with an efficiency of > 60%, with little differential dephasing. Combined with C-H dipolar dephasing, the CSA filter can identify ketal (unprotonated O-C-O) carbons unambiguously for the first time. Conversely, after short cross polarization and the CSA filter, O-CH-O (acetal) carbon signals are observed selectively. The methods are demonstrated on various model compounds and applied to a humic acid.     

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.     

Recoupled polarization transfer heteronuclear 1H-13C multiple-quantum correlation in solids under ultra-fast MAS.

A new approach for high-resolution solid-state heteronuclear multiple-quantum MAS NMR spectroscopy of dipolar-coupled spin-12 nuclei is introduced. The method is a heteronuclear chemical shift correlation technique of abundant spins, like 1H with rare spins, like 13C in natural abundance. High resolution is provided by ultra-fast MAS and high magnetic fields, high sensitivity being ensured by a direct polarization transfer from the abundant protons to 13C. In a rotor-synchronized variant, the method can be used to probe heteronuclear through-space proximities, while the heteronuclear dipolar coupling constant can quantitatively be determined by measuring multiple-quantum spinning-sideband patterns. By means of recoupling, even weak heteronuclear dipolar interactions are accessible. The capabilities of the technique are demonstrated by measurements on crystalline L-tyrosine hydrochloride salt.     

Studies on the Spin-Lattice Relaxation Times of the Carbons of the Investigational Antileukemic Drug and Enzyme Inhibitor Methylglyoxal Bis(Amidino-Hydrazone) ['Methylglyoxal BIS(Guanylhydrazone)'] and Its Dialkylglyoxal Analogs

The first study on the ¹³C relaxation times of bis(amidinohydrazones) is reported. The spin-lattice relaxation times (T₁) of the carbons of the free bases of methylglyoxal bis(amidinohydrazone) (MGBG) and of four dialkylglyoxal analogs thereof were determined with the aid of the inversion recovery method and using dimethyl sulfoxide as the solvent. In the series of compounds studied, one of the side chains was always a methyl group, while the other one was altered (hydrogen, methyl, ethyl, propyl, butyl). Remarkable differences were found to exist between the T₁ values of the various carbons within each molecule. The T₁ values were in the range 1.5 - 2 s for methyl carbons, 0.16 - 1.9 s for carbons of longer alkyl groups, 4.3 - 7.0 s for unprotonated carbons of the glyoxal moiety, 0.57 s for the protonated glyoxal carbon of MGBG, and 2.6 - 3.1 s for guanidino carbons. The bulk of the differences are explainable by assuming that the major relaxation mechanism for the protonated carbons is dipolar relaxation. In alkyl side chains, the T₁ values increased in a very regular fashion down the chain. This effect made possible the assignment of two previously unassigned carbon resonances of the butyl group of BMGBG. T₁ studies thus offer a facile and reliable method for the assignment of side-chain carbon resonances of bis(amidinohydrazones). Further, T₁ measurements were found to offer a very good method for the individual assignment of the glyoxal carbons of unsymmetrical congeners, whose assignment has so far constituted a problem. The method, based on the finding that the one of the carbons bonded to the shorter alkyl chain has a longer relaxation time than does the other one, made possible the unambiguous assignment of several previously unassigned carbon resonances. The results obtained also offer a reliable method for unambiguously distinguishing between the resonances of glyoxal carbons and guanidino carbons that have been difficult to distinguish from each other because the separation of their chemical shifts is often extremely small. Correlations observed between T₁ values and the degree of alkyl substitution in the molecule are discussed, as are also possible relaxation mechanisms. Somewhat unexpectedly, the results obtained suggest that dipolar relaxation through the hydrogens of neighboring carbon atoms may to a significant extent contribute to the relaxation of some unprotonated carbons in bis(amidinohydrazones).     

Inverse polarization transfer for detecting in vivo 13C magnetization transfer effect of specific enzyme reactions in 1H spectra

The wide chemical shift dispersion and long T(1) of (13)C have allowed determination of in vivo magnetization transfer effects caused by aspartate aminotransferase and lactate dehydrogenase reactions using (13)C magnetic resonance spectroscopy. In this report, we demonstrate that these effects can be observed in the proton spectra by transferring the equilibrium magnetization of (13)C via the one-bond scalar coupling between (13)C and (1)H using an inverse insensitive nuclei enhanced by polarization transfer-based heteronuclear polarization transfer method. This inverse method allows a combination of the advantages of the long (13)C T(1) for maximum magnetization transfer and the high sensitivity of proton detection. The feasibility of this in vivo inverse polarization transfer approach was evaluated for detecting the (13)C magnetization transfer effect of aspartate aminotransferase and lactate dehydrogenase reactions from a 72.5-microl voxel in the rat brain at 11.7 T.     

A model for establishing the ultimate enhancements (A∞) in the low to high magnetic field transfer dynamic nuclear polarization experiment

In a preliminary report, we have demonstrated transfer of a flowing bolus enhanced in low magnetic fields (e.g., 0.33 T) with dynamic nuclear polarization (DNP), but monitored in a high magnetic field (4.7 T). The advantages of the high magnetic field monitoring approach include: 1) greater chemical shift dispersion, and 2) improved signal strength in comparison with static low field DNP experiments. In the present study, a model is developed to predict ultimate DNP enhancements (A_∞) in this experiment for flow liquid/liquid intermolecular transfer (L²IT). L²IT¹H and¹³C data is obtained for benzene and chloroform in order to test the validity of the model. The ultimate¹H and¹³C DNP enhancements obtained for benzene/TEMPO are ?150 and ?220, respectively. For a chloroform/TEMPO (L²IT) sample, the ultimate enhancements are close to the¹H dipolar (?330) and the¹³C scalar (+2660) limit, respectively. In the latter case, the observed¹³C DNP enhancement exceeds the thermal Boltzmann magnetization at 4.7 T by a factor of 21. For a 1-chlorobutane/TEMPO sample selective enhancements were observed at different sites in the molecule. For example, the C-1 carbon exhibits a large scalar enhancement, whereas, the other carbons exhibit dipolar enhancements. Data illustrating the importance of three-spin effects in¹³C DNP studies is also presented. Alternative methods of sample transfer from the low to high magnetic field are also discussed.     

1H and 13C NMR studies of glycine in anisotropic media: double-quantum transitions and the effects of chiral interactions

The (1)H NMR spectrum of glycine in stretched gelatin gel and in cromolyn liquid crystal displays a well-resolved doublet due to (1)H-(1)H dipolar interaction. Multiple spectra were obtained within a wide range of offset frequencies of partially saturating radio-frequency (RF) radiation to generate steady-state irradiation envelopes or z-spectra of glycine. Maximal suppression of the doublet occurred when the irradiation was applied exactly at the centre frequency, between the two glycine peaks. This phenomenon is due to double-quantum transitions and is similar to our previous work on quadrupolar nuclei (2)H (HDO) and (23)Na(+). When the (13)C isotopomer glycine-2-(13)C was used, the same effect was found in twice, split by (1)J(CH)+2D(CH). Additional signals in (1)H and (13)C NMR due to prochiral-chiral interactions were found when glycine-2-(13)C was dissolved in chiral anisotropic gelatin and κ-carrageenan gels. The NMR spectra were successfully simulated assuming a (2)J(HH) coupling constant of -16.5Hz and two distinct dipolar coupling constants for the -(13)CH(2)- group (D(C,HA), and D(C,HB)).     

Geometry of multiple-spin systems as reflected in 13C-{1H} dipolar spectra measured at Lee-Goldburg cross-polarization

Theoretical calculation and analysis of (13)C-{(1)H} dipolar spectra of small-size spin clusters is presented. Dipolar spectra simulated using the time-independent average Hamiltonian are compared with the dipolar profiles obtained by 2D and 3D (1)H-(13)C correlation experiments employing Lee-Goldburg off-resonance cross-polarization (LG-CP). It is demonstrated that the structural parameters such as interatomic distances as well as mutual orientation of internuclear vectors can be derived from the dipolar profiles of simple spin clusters. Simplified analysis of the dipolar spectra based on isolated-like spin-pair approach can be used only if interacting spin cluster is reduced to the three-spin system in which the angle between both internuclear vectors ranges from 45 degrees to 135 degrees . For other local arrangements of spin systems the produced dipolar spectra must be analyzed with high caution. Contributions of all interacting spins to dipolar evolution of (13)C magnetization are mutually mixed and cannot be easily separated. However, simplification of the dipolar spectra is achieved by selective excitation. Enhanced selectivity of LG-CP transfer due to the initial (1)H chemical-shift-evolution period makes it possible to construct the dipolar spectra from (1)H-(13)C cross-peak intensities for every detected (1)H-(13)C spin-pair. Consequently, isolated-like spin pair evolution of the detected (1)H-(13)C coherence dominates to the resulting dipolar profile, while the influence of other interacting spins is suppressed. However, this suppression is not quite complete and analysis of the selective dipolar spectra based on isolated-like spin-pair approach cannot be used generally. Especially evolution of long-range (1)H-(13)C coherence is still significantly affected by spin states of other coupled hydrogen atoms.     

Measurement of long range H,C couplings in natural products in orienting media: a tool for structure elucidation of natural products

In this paper we show that water insoluble compounds dissolved in poly-gamma-benzyl-glutamate are amenable to the measurement of a number of homo- and heteronuclear dipolar couplings. The sensitivity and experimental precision of dipolar couplings are sufficient to obtain a good match with the structure. In order to achieve the necessary precision for H,C dipolar couplings between protons and carbons that are not directly bound a new method for the measurement of heteronuclear long range couplings is introduced that allows a one-parameter fit to a HSQC-based experiment as reference experiment. The methodology is applied to menthol (1R, 3S, 4R).     

One- and two-dimensional 13C-1H/15N- 1H dipolar correlation experiments under fast magic-angle spinning for determining the peptide dihedral angle phi

A recently proposed 13C-1H recoupling sequence operative under fast magic-angle spinning (MAS) [K. Takegoshi, T. Terao, Solid State Nucl. Magn. Reson. 13 (1999) 203-212.] is applied to observe 13C-1H and 15N-1H dipolar powder patterns in the IH-15N- 3C- H system of a peptide bond. Both patterns are correlated by 15N-to-13C cross polarization to observe one- or two-dimensional (1D or 2D) correlation spectra, which can be simulated by using a simple analytical expression to determine the H-N-C-H dihedral angle. The 1D and 2D experiments were applied to N-acetyl[1,2-13C,15N] DL-valine, and the peptide q angle was determined with high precision by the 2D experiment to be +/- 155.0 degrees +/- 1.2 degrees. The positive one is in good agreement with the X-ray value of 154 degrees +/- 5 degrees. The 1D experiment provided the value of phi = +/- 156.0 degrees +/- 0.8 degrees.     

NMR determination of the torsion angle psi in alpha-helical peptides and proteins: the HCCN dipolar correlation experiment

Several existing methods permit measurement of the torsion angles phi, psi and chi in peptides and proteins with solid-state MAS NMR experiments. Currently, however, there is not an approach that is applicable to measurement of psi in the angular range -20 degree to -70 degree, commonly found in alpha-helical structures. Accordingly, we have developed a HCCN dipolar correlation MAS experiment that is sensitive and accurate in this regime. An initial REDOR driven (13)C'--(15)N dipolar evolution period is followed by the C' to C(alpha) polarization transfer and by Lee--Goldburg cross polarization recoupling of the (13)C(alpha)(1)H dipolar interaction. The difference between the effective (13)C(1)H and (13)C(15)N dipolar interaction strengths is balanced out by incrementing the (13)C--(15)N dipolar evolution period in steps that are a factor of R(R approximately omega(CH)/omega(CN)) larger than the (13)C--(1)H steps. The resulting dephasing curves are sensitive to variations in psi in the angular region associated with alpha-helical secondary structure. To demonstrate the validity of the technique, we apply it to N-formyl-[U-(13)C,(15)N] Met-Leu-Phe-OH (MLF). The value of psi extracted is consistent with the previous NMR measurements and close to that reported in diffraction studies for the methyl ester of MLF, N-formyl-[U-(13)C,(15)N]Met-Leu-Phe-OMe.     

2D(13)C-(13)C MAS NMR correlation spectroscopy with mixing by true (1)H spin diffusion reveals long-range intermolecular distance restraints in ultra high magnetic field

An improved 2D (13)C-(13)C CP(3) MAS NMR correlation experiment with mixing by true (1)H spin diffusion is presented. With CP(3), correlations can be detected over a much longer range than with direct (1)H-(13)C or (13)C-(13)C dipolar recoupling. The experiment employs a (1)H spin diffusion mixing period tau(m) sandwiched between two cross-polarization periods. An optimized CP(3) sequence for measuring polarization transfer on a length scale between 0.3 and 1.0 nm using short mixing times of 0.1 ms < tau(m) < 1 ms is presented. For such a short tau(m), cross talk from residual transverse magnetization of the donating nuclear species after a CP can be suppressed by extended phase cycling. The utility of the experiment for genuine structure determination is demonstrated using a self-aggregated Chl a/H(2)O sample. The number of intramolecular cross-peaks increases for longer mixing times and this obscures the intermolecular transfer events. Hence, the experiment will be useful for short mixing times only. For a short tau(m) = 0.1 ms, intermolecular correlations are detected between the ends of phytyl tails and ring carbons of neighboring Chl a molecules in the aggregate. In this way the model for the structure, with stacks of Chl a that are arranged back to back with interdigitating phytyl chains stretched between two bilayers, is validated.     

Heteronuclear Double-Quantum MAS NMR Spectroscopy in Dipolar Solids

A new pulse sequence for high-resolution solid-state heteronuclear double-quantum MAS NMR spectroscopy of dipolar-coupled spin-12 nuclei is introduced. It is based on the five-pulse sequence known from solution-state NMR, which is here applied synchronously to both spin species. The heteronuclear double-quantum (HeDQ) spinning-sideband patterns produced by this experiment are shown to be sensitive to the heteronuclear distance, as well as the relative orientations of the chemical-shift and dipolar tensors. In particular, it is shown that the HeDQ patterns exhibit an enhanced sensitivity to the chemical shielding tensors as compared with the single-quantum spinning-sideband patterns. The detection of HeDQ patterns via the I and S spins is discussed. The isolated (13)C-(1)H spin pair in deuterated ammonium formate with (13)C in natural abundance was chosen as a model system, and the perturbing influence of dipolar couplings to surrounding protons on the (13)C-(1)H DQ coherence is discussed. The pulse sequence can also be used as a heteronuclear double-quantum filter, hence providing information about heteronuclear couplings, and thus allowing the differentiation of quaternary and CH(n) bonded carbons. The elucidation of (13)C-(1)H dipolar proximities is presented for a sample of bisphenol A polycarbonate with (13)C in natural abundance, recorded with a broadband version of the synchronized five-pulse sequence.     

Dynamic nuclear polarization at 9T using a novel 250 GHz gyrotron microwave source. 2003

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 9 T (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 20 K; in addition, we have observed significant enhancements in ¹⁵N spectra of unoriented pf1-bacteriophage. Finally, enhancements of ∼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.     

Kinetics of NMR spin-lock polarization transfer in crystalline glycine and spin-lattice relaxation of amino acids

We report data determined from proton-carbon polarization-transfer kinetics at 23 degrees C for six common solid amino acids. Proton spin-lattice relaxation times in the rotating frame, T(1rhoH), for alpha-glycine, alanine, cysteine, leucine, isoleucine, and valine determined from the long-time decay of the carbon magnetization indicate that the presence of a mobile entity such as a methyl group shortens T(1rhoH) to a few milliseconds. Polarization transfer between protons and carbons in polycrystalline alpha-glycine is analyzed and compared to theoretical models, two of which account for the variation of polarization-transfer rate with orientation of the dipole-dipole vector in the magnetic field. A generalization of a model proposed by Mueller et al. (Phys. Rev. Lett. 32 (1974) 1402) reproduces the observed polarization transfer in alpha-glycine with reasonable accuracy, showing that the early time development reflects orientational variation of dipolar oscillations.