1H Decoupling Research Articles

Fullerene as a Standard Sample for Adjusting the NMR Spectral Resolution in Multiple-Quantum Magic-Angle-Spinning Experiments

A method using fullerene for adjusting the NMR spectral resolution for multiple quantum magic angle spinning (MQMAS) experiments is proposed. To observe its (13)C MAS signal, it is not necessary to apply (1)H decoupling, unavailable with single-resonance MQMAS probes. Although (13)C T(1) of fullerene is rather long, a recycle time of 5 s in shimming yields its signal with sufficiently high sensitivity if setting the appropriate Ernst angles corresponding to magnetic fields. It is demonstrated that so-adjusted high resolution is reflected in the (87)Rb MQMAS spectra of RbNO(3).

Differential Line Broadening in MAS Solid-State NMR due to Dynamic Interference

Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond-microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NH(alpha/beta) multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N-H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra were obtained. We expect that this approach which requires intense deuteration will have a significant impact on the quality and the rate at which solid-state NMR spectroscopic investigations will emerge in the future.

Broadband homonuclear chemical shift correlation at high MAS frequencies: a study of tanh/tan adiabatic RF pulse schemes without $${^{{\bf 1}}\hbox{{\bf H}}}$$ decoupling during mixing

At high magic angle spinning (MAS) frequencies the potential of tanh/tan adiabatic RF pulse schemes for 13C chemical shift correlation without 1H decoupling during mixing has been evaluated. It is shown via numerical simulations that a continuous train of adiabatic 13C inversion pulses applied at high RF field strengths leads to efficient broadband heteronuclear decoupling. It is demonstrated that this can be exploited effectively for generating through-bond and through-space, including double-quantum, correlation spectra of biological systems at high magnetic fields and spinning speeds with no 1H decoupling applied during the mixing period. Experiments carried out on a polycrystalline sample of histidine clearly suggest that an improved signal to noise ratio can be realised by eliminating 1H decoupling during mixing.

Low-load rotor-synchronised Hahn-echo pulse train (RS-HEPT)1H decoupling in solid-state NMR: factors affecting MAS spin-echo dephasing times

Transverse dephasing times T(2)' in spin-echo MAS NMR using rotor-synchronised Hahn-echo pulse-train (RS-HEPT) low-load (1)H decoupling are evaluated. Experiments were performed at 300 and 600 MHz for (13)CH-labelled L-alanine and (15)NH(delta)-labelled L-histidine.HCl.H(2)O, together with SPINEVOLUTION simulations for a ten-spin system representing the crystal structure environment of the (13)CH carbon in L-alanine. For 30 kHz MAS and nu(1)((1)H) = 100 kHz at 300 MHz, a RS-HEPT T(2)' value of 17 +/- 1 ms was obtained for (13)CH-labelled L-alanine which is approximately 50% of the XiX T(2)' value of 33 +/- 2 ms. Optimum RS-HEPT decoupling performance is observed for a relative phase of alternate RS-HEPT pi-pulses, Deltaphi = phi'- phi, between 40 and 60 degrees . For experiments at 600 MHz and 30 kHz MAS with (13)CH-labelled L-alanine, the best RS-HEPT (nu(1)((1)H) = 100 kHz) T(2)' value was 3 times longer than that observed for low-power continuously applied sequences with nu(1)((1)H) < or =40 kHz, i.e. corresponding to the same average power dissipated in the probe. A marked improvement in RS-HEPT (1)H decoupling is observed for increasing MAS frequency: at 55.6 kHz MAS, a best RS-HEPT T(2)' value of 34 +/- 5 ms was recorded for (13)CH-labelled L-alanine. Much improved RS-HEPT broadband performance was also observed at 55.6 kHz MAS as compared to 30 kHz MAS.

A Morphological Study of Poly[Bis(Trifluoroethoxy)Phosphazene] Using Solid-State NMR: Introducing Domain Selective 1H and 19F Decoupled 13C MAS NMR

Using solid-state NMR methods the morphological behavior of poly[bis(trifluoroethoxy)phosphazene] was studied, employing four nuclei of interest – 1H, 19F, 31P and 13C. Measurements on all four nuclei support that at ambient temperature the crystalline and amorphous phases coexist. Variable temperature studies showed that above T(1) = 90° only a single highly mobile phase exists, which is presumed to be the 2D mesophase. All four nuclei showed that when heat cycling the polymer, repeatedly above T(1), an increase in crystallinity occurs with each cycle. For the first time 13C MAS NMR spectra, using high power 19F and 1H decoupling, were obtained, which exhibited the same behaviour domain. Filtered 13C{1H,19F} MAS spectra containing signal from the crystalline domain using the discrimination induced by variable amplitude minipulses (DIVAM) sequence were measured. Heat treated and solvent cast material showed differences in these 13C spectra, that were consistent with a decrease in backbone conformations upon heating, suggesting an increase in the extended chain form corresponding to the γ form. Analogous sensitivity to variations in crystal phase composition has not been seen previously using 1H, 19F and 31P methods, emphasizing the importance of 13C MAS methods to morphological studies of phosphazenes.

Unambiguous Determination of the Ionization State of a Glycoside Hydrolase Active Site Lysine by 1H−15N Heteronuclear Correlation Spectroscopy

We have investigated the lysine side chain amines in the 34 kDa catalytic domain from Cellulomonas fimi beta-(1,4)-glycosidase Cex (or CfXyn10A) using 1H-detected 15N heteronuclear correlation NMR spectroscopy. Signals from the 1Hzeta ( approximately 8 ppm) and 15Nzeta ( approximately 35 ppm) of Lys302 in the unmodified enzyme and Lys47 in a trapped cellobiosyl-enzyme intermediate were detected in a 1H-15N HMQC spectrum (pH 6.5 and 30 degrees C). The amine of Lys302 forms a buried ion pair, and that of Lys47 is hydrogen bonded to the cellobioside. Both lysines are positively charged, as unambiguously demonstrated by the splitting of their 15Nzeta signals into quartets (|1JNH| approximately 75 Hz) in a 1H-15N HSQC spectrum recorded without 1H decoupling during 15N evolution. Qualitative insights into the dynamic properties of these lysines are also provided by the deviations of their quartet intensity ratios from that of approximately 3:1:1:3 expected for a highly mobile amine. On the basis of the observed ratios of approximately 1:1:1:1 for the quartet of Lys302 and approximately 0.5:1:1:0.5 for Lys47, the amine of the latter active site residue is most rigidly positioned. Signals from at least 8 and 10 additional positively charged, mobile amines in Cex were observed at 10 degrees C and pH 6.5 and 5.6, respectively. By using conditions of reduced temperature, slightly acidic pH, and low general base concentrations, as well as water flipback pulses to minimize the effects of hydrogen exchange, 1H-15N correlation experiments provide a sensitive route to directly investigate the charge states and dynamic properties of the N-terminal and side chain amines in proteins and protein complexes.

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 ⩾20 kHz) enhances resolution/sensitivity in 13C SSNMR for paramagnetic complexes [Y. Ishii, S. Chimon, N.P. Wickramasinghe, A new approach in 1D and 2D 13C 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 13C 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 13C signal is observed under VFMAS without 1H decoupling. The resulting low duty factor permits rapid signal accumulation without probe arcing at recycle times (∼3 ms/scan) matched to short 1H T 1 values of small paramagnetic systems (∼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 13C MAS spectrum under moderate MAS at 5 kHz. The applicability of this approach was also demonstrated for a more challenging system, Mn(acac) 3, for which 13C and 1H 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 13CH, 13CH 2, 13CH 3, 13 CO 2 - groups in 1D experiments for Cu( dl-Ala) 2 and Cu(Gly) 2. Applications of this technique to 2D 13C/ 1H correlation NMR under VFMAS yielded reliable assignments of 1H resonances as well as 13C resonances for Cu( dl-Ala) 2 and Mn(acac) 3. Quantitative analysis of cross-peak intensities in 2D 13C/ 1H correlation NMR spectra of Cu( dl-Ala) 2 provided distance information between non-bonded 13C– 1H pairs in the paramagnetic system.

Improvement of the inverse-gated-decoupling sequence for a faster quantitative analysis of various samples by 13C NMR spectroscopy

The inverse-gated-decoupling sequence enables quantitative 1H decoupled 13C spectra to be obtained. We modified this sequence so as to obtain the same result in less time for molecules containing carbons with various relaxation properties. For that, we determined the optimal 13C longitudinal-magnetization initial value for a faster relaxation while 1H decoupler is stopped. This value can be calculated precisely via the nuclear Overhauser effects, the longitudinal relaxation times, together with the determination of the relaxation rate constants of carbons while 1H are out of equilibrium. A supplementary delay of 1H decoupling and/or a series of selective pulses applied at the beginning of the recovery delay allow an acceleration of 13C longitudinal relaxation. We applied this method to the molecule of vanillin. The simultaneous quantification of all carbons was carried out with a recovery delay divided by two compared to the usual sequence.

Enhancing resolution and sensitivity of 17O solid-state NMR through combining double rotation, 1H decoupling and satellite modulation for biomolecular applications

17O solid-state NMR is a highly sensitive probe of structural detail of organic solids but improvements in sensitivity and resolution are crucial for it to be applied to larger biological molecules. Here it is shown that high resolution (∼1 ppm) and significant signal enhancement can be achieved by combining 1H decoupled double rotation (DOR), which narrows the lines by a factor of ∼100 compared to conventional magic angle spinning, and manipulation of the satellite transition populations to transfer magnetisation to the central transition, which produces a signal enhancement of ∼2.

Broadband Homonuclear Correlation Spectroscopy at High Magnetic Fields and MAS Frequencies

We present a new homonuclear recoupling sequence, CMAR, that allows observation of 2D 13C-13C correlation spectra at high magnetic fields and MAS frequencies (10-30 kHz). The main advantages of the sequence are that it provides efficient, broadband dipolar recoupling and concurrently decouples the 1H spins from the 13C's. Thus, no additional 1H decoupling is required during the mixing period, thereby significantly reducing the radio frequency power requirements for the experiment. Thus, CMAR significantly extends the range of applicability of the usual homonuclear recoupling techniques and should be of major interest for structure determinations of biomolecules at high magnetic fields.

Investigating brain metabolism at high fields using localized 13C NMR spectroscopy without 1H decoupling

Most in vivo 13C NMR spectroscopy studies in the brain have been performed using 1H decoupling during acquisition. Decoupling imposes significant constraints on the experimental setup (particularly for human studies at high magnetic field) in order to stay within safety limits for power deposition. We show here that incorporation of the 13C label from 13C-labeled glucose into brain amino acids can be monitored accurately using localized 13C NMR spectroscopy without the application of 1H decoupling. Using LCModel quantification with prior knowledge of one-bond and multiple-bond J(CH) coupling constants, the uncertainty on metabolites concentrations was only 35% to 91% higher (depending on the carbon resonance of interest) in undecoupled spectra compared to decoupled spectra in the rat brain at 9.4 Tesla. Although less sensitive, 13C NMR without decoupling dramatically reduces experimental constraints on coil setup and pulse sequence design required to keep power deposition within safety guidelines. This opens the prospect of safely measuring 13C NMR spectra in humans at varied brain locations (not only the occipital lobe) and at very high magnetic fields above 4 Tesla.

Efficient spin–spin scalar coupling mediated C-13 homonuclear polarization transfer in biological solids without proton decoupling

We demonstrate that an efficient C′↔C α polarization transfer based on J-coupling can be realized under fast magic-angle spinning (MAS) condition without 1H decoupling. Experimental results are presented for model crystalline compounds as well as a non-crystalline 17-residue polypeptide MB( i+4)EK. Measurements on MB( i+4)EK demonstrate that 53% of the initial C′ polarization was transferred to the cross peaks at 7.05 T under 25 kHz MAS spinning.

A solid-state 13C NMR analysis of molecular dynamics in aramide polymers

The local dynamics of aromatic cores was analyzed for a homologous series of polyamides in the solid phase incorporating phenyl, biphenyl and naphthyl groups. Preliminary wide-line and spin-relaxation variable-temperature 1H NMR measurements revealed the presence of thermally activated molecular motions for each polymer studied. A number of 13C NMR experiments were then implemented to further clarify the nature and extent of such motions. These included 1H– 13C 2D separate-local-field measurements, whose line shapes revealed that motions involved for all cases a superposition of states. These could in principle be associated with rigid and mobile populations in these semi-crystalline aramides, a model that yielded a proper description of the spectra at all temperatures. To further probe this model the relaxation behavior of the aramides’ 13C spins was monitored in the rotating frame as a function of temperature, in both the presence and absence of homonuclear 1H– 1H decoupling. The variations observed in these measurements evidenced a thermally activated, relatively broad distribution of motional rates in the polymers. Editing the 2D local-field data according to the 13C relaxation also supported this heterogeneous dynamic model. The mechanism underlying this behavior and implications towards the 13C analysis of motions in aramides in particular and complex polymers in general, is briefly discussed.

Improvement of the inverse-gated-decoupling sequence for a faster quantitative analysis by 13C NMR

Abstract The inverse-gated-decoupling sequence enables quantitative 1H decoupled 13C spectra to be obtained. We modified this sequence so as to obtain the same result in less time. For that, we determined the optimal 13C longitudinal-magnetisation initial value for a faster relaxation while 1H decoupler is stopped. This value can be calculated via the longitudinal relaxation times and the nuclear Overhauser effects. For a given nucleus, a supplementary delay of 1H decoupling and/or a pulse at the beginning of the recovery delay allows an acceleration of the 13C longitudinal relaxation. We checked this result on the molecule of N,N-dimethylacetamide. A simultaneous quantitative analysis of all carbons was carried out with a recovery delay divided by four compared to the usual sequence. To cite this article: P. Giraudeau et al., C. R. Chimie 9 (2006).

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.

Setting up 13C CP/MAS experiments

AbstractThe 13C cross‐polarization (CP) technique combined with magic angle spinning (MAS) has become one of the more commonly performed solid‐state nuclear magnetic resonance (NMR) experiments. The basics of initially setting up the experiment are given and used to illustrate such NMR phenomena as rotational echoes, homogeneous and inhomogeneous interactions, continuous wave 1H decoupling, and coupling of quadrupolar 14N nuclei to 13C nuclei. The polarization transfer from the protons to the carbons is described briefly with the usual thermodynamic and quantum mechanical models. The setup and use of the experiment for routine analyses are discussed. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson 22A: 37–49, 2004.

Resolution of 13C– 19F interactions in the 13C NMR of spinning solids and liquid crystals

Anomalous line-broadenings of carbon resonances close to 19F have commonly been reported in the 13C NMR of liquid crystals and solids. We have previously shown that these effects in static liquid-crystal samples are related to the difficulty of 1H decoupling in the presence of strong 1H– 19F dipolar interactions. We here extend this work to spinning samples (both liquid crystals and solids). A number of different line-broadening mechanisms are elucidated: analogous decoupling effects, magic angle misset, and 19F lifetime-broadening. In relatively mobile systems, such as liquid crystals or soft solids, the limiting factor on 13C resolution (and the ability to directly quantify the 13C– 19F interactions) is found to be the efficiency and robustness of the 1H-decoupling. In rigid solids, the lifetime of the 19F spin-states is found to be an additional critical factor.

Sensitivity-enhanced quantitative 13C NMR spectroscopy via cancellation of 1J(CH) dependence in DEPT polarization transfers.

A scheme has been developed to eliminate virtually all signal intensity dependence on 1JCH in polarization transfers between 1H and 13C nuclei, reducing differences in signal intensity to only 1.5% over the entire natural 1JCH range. The scheme relies on the summation of time-domain data acquired with four suitably selected Delta delays so that the J dependence is essentially canceled in the final, signal-averaged free-induction decay. These Delta delays have been incorporated into the DEPT pulse sequence to create sensitivity-enhanced experiments for collecting quantitative 13C{1H} spectra. Four experiments, each with unique read pulse angles, give quantitative spectra with 200-300% more sensitivity than conventional 13C spectra acquired with inverse-gated 1H decoupling. The experiments are ideal for recording spectra with improved quantitative information or for substantially reducing the long acquisition times indicative of quantitative 13C experiments. The ability of the experiments to provide quantitative spectra was demonstrated with a simple ethylbenzene solution, however, they can easily be adapted to various applications for analysis of complex mixtures.

NMR and modelling studies of disaccharide conformation

Long-range heteronuclear coupling constants were measured across the glycosidic linkages for a series of eight α- or β-linked disaccharides in aqueous solution. Multiple 13C site-selective excitation experiments using 1H decoupling in conjunction with pulsed field gradient-enhanced spectroscopy were used to determine 3 J C,H values. These were subsequently compared with the respective couplings calculated, using a Karplus relationship, from molecular dynamics simulations with the explicit inclusion of water.

A new approach in 1D and 2D 13C high-resolution solid-state NMR spectroscopy of paramagnetic organometallic complexes by very fast magic-angle spinning.

Novel 1D and multidimensional solid-state NMR (SSNMR) methods using very fast magic-angle spinning (VFMAS) (spinning speed > 20 kHz) for performing 13C high-resolution SSNMR of paramagnetic organometallic complexes are discussed. VFMAS removes a majority of 13C-1H and 1H-1H dipolar couplings, which are often difficult to remove by RF pulse techniques in paramagnetic complexes because of large paramagnetic shifts. In the first systematic approach using the unique feature of VFMAS for paramagnetic complexes, we demonstrate a means of obtaining well-resolved 1D and multidimensional 13C SSNMR spectra, sensitivity enhancements via cross polarization, and signal assignments, and applications of dipolar recoupling methods for nonlabeled paramagnetic organometallic complexes of moderate paramagnetic shifts ( approximately 800 ppm). Experimental results for powder samples of small nonlabeled coordination complexes at 1H frequencies of 400.2-400.3 MHz show that highly resolved 13C SSNMR spectra can be obtained under VFMAS, without requirements of 1H decoupling. Sensitivity enhancement in 13C SSNMR via cross polarization from 1H spins was demonstrated with an amplitude-sweep high-power CP sequence using strong RF fields ( approximately 100 kHz) available in the VFMAS probe. 13C CPMAS spectra of nonlabeled Cu(II)(dl-alanine)2.(H2O) and V(III)(acetylacetonate)3 (V(acac)3) show that it is possible to obtain high-resolution spectra for a small quantity ( approximately 15 mg) of nonlabeled paramagnetic organometal complexes within a few minutes under VFMAS. Experiments on Cu(II)(dl-alanine)2.(H2O) demonstrated that 1H-13C dipolar recoupling for paramagnetic organometal complexes can be performed under VFMAS by application of rotor-synchronous pi-pulses to 1H and 13C spins. The results also showed that signal assignments for 13CH, 13CH3, and 13CO groups in paramagnetic complexes are possible on the basis of the amount of 13C-1H dipolar dephasing induced by dipolar recoupling. Furthermore, the experimental 2D 13C/1H chemical-shift correlation NMR spectrum obtained for nonlabeled V(acac)3 exhibits well-resolved lines, which overlap in 1D 13C and 1H spectra. Signals for different chemical groups in the 2D spectrum are distinguished by the 13C-1H dipolar dephasing method combined with the 2D 13C/1H correlation NMR. The assignments offer information on the existence of nonequivalent ligands in the coordination complex in solids, without requiring a single-crystal sample.