Complete Dipolar Decoupling ofC and Its Use in Two-Dimensional Double-Quantum Solid-State NMR for Determining Polymer Conformations

A multiple-pulse technique for complete dipolar decoupling of directly bonded¹³C-labeled sites is described. It achieves significant spectral simplifications in a recently introduced two-dimensional double-quantum solid-state NMR experiment for determining torsion angles. Both homonuclear and heteronuclear dipolar couplings are removed by combining a¹³C multiple-pulse sequence with continuous-wave irradiation on the protons. The¹³C sequence has a fundamental 10-pulse cycle which is a significantly modified magic-sandwich-echo sequence. The crucial heteronuclear decoupling is achieved by breaking the 360° “inner” pulses in the magic sandwich into 90° pulses and spacing them by¹H 360° pulse lengths. Spectral artifacts typical of multiple-pulse sequences are eliminated by phase shifts between cycles. In contrast to many other multiple-pulse decoupling sequences, the long window in the cycle is the dwell time and can be longer than the inverse dipolar coupling, which makes the sequence practical for direct detection even with long pulse ring-down times. A modification of the sequence to scale the chemical shift and increase the effective spectral width is also presented. The 1D and double-quantum 2D experiments are demonstrated on polyethylene with 4%¹³C–¹³C spin pairs. The potential of this approach for distinguishing segmental conformations is illustrated by spectral simulations of the two-dimensional ridge patterns that correlate double-quantum and single-quantum chemical-shift anisotropies.     

A New Two-Dimensional Pulse Sequence for T2* Measurements of Protons in C Isotopomers

A new two-dimensional pulse sequence for T₂* measurement of protons directly coupled to ¹³C spins is proposed. The sequence measures the tranverse relaxation time of heteronuclear proton single-quantum coherence under conditions of free precession and is therefore well suited to evaluate relaxation losses of proton magnetization during preparation delays of heteronuclear pulse experiments in analytical NMR. The relevant part of the pulse sequence can be inserted as a “building block” into any direct or inverse detecting H,C correlation pulse sequence if proton spin–spin relaxation is to be investigated. In this contribution, the building block is inserted into a HETCOR as well as into a HMQC pulse sequence. Experimental results for the HETCOR-based sequence are given.     

Cooling overall spin temperature: protein NMR experiments optimized for longitudinal relaxation effects

In experiments performed on protonated proteins at high fields, 80% of the NMR spectrometer time is spent waiting for the ¹H atoms to recover their polarization after recording the free induction decay. Selective excitation of a fraction of the protons in a large molecule has previously been shown to lead to faster longitudinal relaxation for the selected protons [K. Pervushin, B. Vögeli, A. Eletsky, Longitudinal ¹H relaxation optimization in TROSY NMR spectroscopy, J. Am. Chem. Soc. 124 (2002) 12898–12902; P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015; H.S. Attreya, T. Szyperski, G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment, Proc. Natl. Acad. Sci. USA 101 (2004) 9642–9647]. The pool of non-selected protons acts as a “thermal bath” and spin-diffusion processes (“flip-flop” transitions) channel the excess energy from the excited pool to the non-selected protons in regions of the molecule where other relaxation processes can dissipate the excess energy. We present here a sensitivity enhanced HSQC sequence (COST-HSQC), based on one selective E-BURP pulse, which can be used on protonated ¹⁵N enriched proteins (with or without ¹³C isotopic enrichment). This experiment is compared to a gradient sensitivity enhanced HSQC with a water flip-back pulse (the water flip-back pulse quenches the spin diffusion between ¹H^N and ¹H^α spins). This experiment is shown to have significant advantages in some circumstances. Some observed limitations, namely sample overheating with short recovery delays and complex longitudinal relaxation behaviour are discussed and analysed.     

Reconstruction of randomly under-sampled spectra for in vivo 13C magnetic resonance spectroscopy

PurposeOver the past decade, many techniques have been developed to reduce radiofrequency (RF) power deposition associated with proton decoupling in in vivo Carbon-13 (¹³C) magnetic resonance spectroscopy (MRS). In this work we propose a new strategy that uses data under-sampling to achieve reduction in RF power deposition.Materials and methodsEssentially, proton decoupling is required only during randomly selected segments of data acquisition. By taking advantage of the sparse spectral pattern of the carboxylic/amide region of in vivo ¹³C spectra of brain, we developed an iterative algorithm to reconstruct spectra from randomly under-sampled data. Fully sampled data were used as references. Reconstructed spectra were compared with the fully sampled references and evaluated using residuals and relative signal intensity errors.ResultsNumerical simulations and in vivo experiments at 7 Tesla demonstrated that this novel decoupling and data processing strategy can effectively reduce decoupling power deposition by greater than 30%.ConclusionThis study proposes and evaluates a novel approach to acquire ¹³C data with reduced proton decoupling power deposition and reconstruct in vivo ¹³C spectra of carboxylic/amide metabolite signals using randomly under-sampled data. Because proton decoupling is not needed over a significant portion of data acquisition, this novel approach can effectively reduce the required decoupling power and thus SAR. It opens the possibility of performing in vivo ¹³C experiments of human brain at very high magnetic fields.     

Fluorine-19 Solid-State NMR Magic-Angle-Turning Experiments Using Multiple-Pulse Homonuclear Decoupling

For compounds giving “crowded” 1-dimensional magic-angle-spinning spectra, information about the local atomic environment in the form of the chemical shift anisotropy (CSA) is sacrificed for high resolution of the less informative isotropic chemical shift. Magic-angle-turning (MAT) NMR pulse sequences preserve the CSA information by correlating it to the isotropic chemical shift in a 2-dimensional experiment. For low natural abundance nuclei such as ¹³C and ¹⁵N and under ¹H heteronuclear dipolar decoupling conditions, the dominant NMR interaction is the chemical shift. For abundant nuclei such as ¹H, ¹⁹F, and ³¹P, the homonuclear dipolar interaction becomes a significant contribution to the observed linewidth in both F₁ and F₂ dimensions. We incorporate MREV8 homonuclear multiple-pulse decoupling sequences into the MAT experiment to give a multiple-pulse MAT (MP-MAT) experiment in which the homonuclear dipolar interaction is suppressed while maintaining the chemical shift information. Extensive use of computer simulation using GAMMA has guided the pulse sequence development. In particular, we show how the MREV8 pulses can be incorporated into a quadrature-detected sequence such as MAT. The MP-MAT technique is demonstrated for a model two-site system containing a mixture of silver trifluoroacetate and calcium difluoride. The resolution in the isotropic evolution dimension is improved by faster sample spinning, shorter MREV8 cycle times in the evolution dimension, and modifications of the MAT component of the pulse sequence.     

柚皮素7-O-葡萄糖苷非对映异构体的NMR波谱分析

二氢黄酮糖苷化后产生的R与S构型非对映异构体在¹H NMR谱上会呈现一些差别,但文献对其差别描述非常有限.为便于利用¹H NMR谱图判断二氢黄酮糖苷的R与S构型非对映异构体,本文首先在植物药皂荚提取物中分离得到一种二氢黄酮苷-柚皮素7-O-葡萄糖苷R与S构型混合物,分析其氘代二甲亚砜（DMSO-d₆）溶液的¹H NMR、¹³C NMR、¹H-¹H COSY、¹H-¹³C HSQC和¹H-¹³C HMBC谱,对其¹H和¹³C NMR谱峰进行了归属;然后,采用手性色谱柱对该混合物进行分离,结合圆二色光谱（CD）技术确定构型;最后,为鉴别R与S构型柚皮素7-O-葡萄糖苷在¹H NMR谱中特征差别谱峰,避免葡萄糖残基质子对二氢黄酮苷元质子化学位移的影响,采集了R与S构型柚皮素7-O-葡萄糖苷及其混合物氘代乙腈（CD₃CN）溶液的NMR谱,结果显示葡萄糖残基端基质子H-1″化学位移差别最为明显,为9.4 Hz;5-位酚羟基质子化学位移差别为5.8 Hz,C环上3个质子化学位移差也较明显．     

Proton-detected heteronuclear single quantum correlation NMR spectroscopy in rigid solids with ultra-fast MAS

In this article, we show the potential for utilizing proton-detected heteronuclear single quantum correlation (HSQC) NMR in rigid solids under ultra-fast magic angle spinning (MAS) conditions. The indirect detection of carbon-13 from coupled neighboring hydrogen nuclei provides a sensitivity enhancement of 3- to 4-fold in crystalline amino acids over direct-detected versions. Furthermore, the sensitivity enhancement is shown to be significantly larger for disordered solids that display inhomogeneously broadened carbon-13 spectra. Latrodectus hesperus (Black Widow) dragline silk is given as an example where the sample is mass-limited and the sensitivity enhancement for the proton-detected experiment is 8- to 13-fold. The ultra-fast MAS proton-detected HSQC solid-state NMR technique has the added advantage that no proton homonuclear decoupling is applied during the experiment. Further, well-resolved, indirectly observed carbon-13 spectra can be obtained in some cases without heteronuclear proton decoupling.     

Application of biselective refocusing soft pulses to the simplification of heteronuclear correlation spectra

The biselective spin echo technique allows the signals of coupled proton pairs to be extracted from crowded liquid state proton NMR spectra. Its use as a preparation sequence in heteronuclear chemical shift correlation experiments requires the removal of the heteronuclear coupling interaction during the biselective echo time. The discrimination between coupled and uncoupled protons signals is achieved by double quantum filtration, which delivers antiphase magnetization states. The latter are not directly compatible with the design of an HSQC-like pulse sequence. The conversion of antiphase to in-phase magnetization states by a second biselective echo sequence solves this problem. The optimization of spin echo delays is also discussed. Lastly, the article presents modified HSQC and HMBC pulses sequences in which information is obtained solely for the biselectively selected proton pairs. A peracetylated trisaccharide was used as a test molecule.     

Three-Dimensional C Shift/H–N Coupling/N Shift Solid-State NMR Correlation Spectroscopy

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly ¹³C- and ¹⁵N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from ¹H to ¹³C and from ¹³C to ¹⁵N with flip-flop (phase and frequency switched) Lee–Goldburg irradiation for both ¹³C homonuclear decoupling and ¹H–¹⁵N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields ¹³C shift/¹H–¹⁵N coupling/¹⁵N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate ¹³C and ¹⁵N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly ¹³C- and ¹⁵N-labeled proteins.     

In Vivo3D LocalizedC Spectroscopy Using Modified INEPT and DEPT

The 3D localized¹³C spectroscopy methods LINEPT and LODEPT, which are modifications of INEPT and DEPT, are proposed. As long as a¹³C inversion pulse (180-degree pulse) is applied at 1/(4J) before the proton echo time in LINEPT and a¹³C excitation pulse (90-degree pulse) is applied at 1/(2J) before the proton echo time in LODEPT, the proton echo time can be set to any value longer than 1/(2J) in LINEPT and longer than 1/Jin LODEPT. As a result, the proton and the¹³C pulses can be applied separately and these proton pulses can be made slice-selective pulses. These localization features of LINEPT and LODEPT were evaluated using a phantom consisting of a cylinder filled with ethanol placed inside another cylinder filled with oil, and localized ethanol spectra could be obtained.In vivo3D localized¹³C spectra from the brain of a monkey could be obtained using decoupled LINEPT, and glutamate C-4 appeared directly after the administration of glucose C-1, followed by the appearance of glutamate C-2, C-3 and glutamine C-2, C-3, C-4.     

Accurate Measurements of Multiple-Bond C–H Coupling Constants from Phase-Sensitive 2D INEPT Spectra

Measurements of multiple-bond ¹³C–¹H coupling constants are of great interest for the assignment of nonprotonated ¹³C resonances and the elucidation of molecular conformation in solution. Usually, the heteronuclear multiple-bond coupling constants were measured either by the J_CH splittings mostly in selective 2D spectra or in 3D spectra, which are time consuming, or by the cross peak intensity analysis in 2D quantitative heteronuclear J correlation spectra (1994, G. Zhu, A. Renwick, and A. Bax, J. Magn. Reson. A 110, 257; 1994, A. Bax, G. W. Vuister, S. Grzesiek, F. Delaglio, A. C. Wang, R. Tschudin, and G. Zhu, Methods Enzymol. 239, 79.), which suffer from the accuracy problem caused by the signal-to-noise ratio and the nonpure absorptive peak patterns. Concerted incrementation of the duration for developing proton antiphase magnetization with respect to carbon-13 and the evolution time for proton chemical shift in different steps in a modified INEPT pulse sequence provides a new method for accurate measurements of heteronuclear multiple-bond coupling constants in a single 2D experiment.     

Signal Enhancement Using 45° Water Flipback for 3D N-Edited ROESY and NOESY HMQC and HSQC

A novel implementation of the water flipback technique employing a 45° flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D ¹⁵N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45° water flipback are presented using both HMQC and HSQC for the ¹⁵N dimension. The 45° flipback pulse is followed by a gradient, a water selective 180° pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t₁ and t₂ evolution periods is suppressed using gradients. Water resonance planes from NOESY–HMQC and NOESY–HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein–water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.     

Selective dephasing of OH and NH proton magnetization based on (1)H chemical-shift anisotropy recoupling

A method for selectively suppressing the signals of OH and NH protons in (1)H combined rotation and multiple-pulse spectroscopy (CRAMPS) and in (1)H-(13)C heteronuclear correlation (HETCOR) solid-state NMR spectra is presented. It permits distinction of overlapping CH and OH/NH proton signals, based on the selective dephasing of the magnetization of OH and NH protons by their relatively large (1)H chemical-shift anisotropies. For NH protons, the (14)N-(1)H dipolar coupling also contributes significantly to this dephasing. The dephasing is achieved by a new combination of heteronuclear recoupling of these anisotropies with (1)H homonuclear dipolar decoupling. Since the 180 degrees pulses traditionally used for heteronuclear dipolar and chemical-shift anisotropy recoupling would result in undesirable homonuclear dephasing of proton magnetization, instead the necessary inversion of the chemical-shift Hamiltonian every half rotation period is achieved by inverting the phases of all the pulses in the HW8 multiple-pulse sequence. In the HETCOR experiments, carefully timed (13)C 180 degrees pulses remove the strong dipolar coupling to the nearby (13)C spin. The suppression of NH and OH peaks is demonstrated on crystalline model compounds. The technique in combination with HETCOR NMR is applied to identify the CONH and NH-CH groups in chitin and to distinguish NH and aromatic proton peaks in a peat humin.     

Transverse Relaxation of Scalar Coupled Protons in Magnetic Resonance of Non-Deuterated Proteins

The transverse relaxation rates R ₂ = 1/T ₂ of protons can be determined by spin-echo sequences with multiple refocusing pulses using moderate radio-frequency field strengths and properly chosen inter-pulse delays so as to suppress echo modulations due to homonuclear scalar couplings. Combination with 2D heteronuclear correlation spectroscopy (HSQC) allows one to measure R ₂ of arbitrary protons attached to nitrogen-15 or carbon-13 nuclei. Decays of six amide protons in the protein Ubiquitin that is nitrogen-15 enriched (but not deuterated) were measured at different temperatures.     

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

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

Selective Correlation of Amide Groups to Glycine Alpha Protons and of Arginine Guanidine Groups to Delta Protons in Proteins by Multiple Quantum Spectroscopy

A new 3D pulse sequence correlates backbone amide proton and nitrogen with alpha proton resonances selectively for glycine residues in a fully doubly labeled (¹⁵N,¹³C) protein. The excitation of multiple quantum coherences provides optimized resolution and sensitivity. Degenerate alpha proton groups can be promptly recognized. Correlation of guanidine NH groups to delta protons of arginine side chains is also obtained.     

Spin-state-selective excitation in selective 1D inverse NMR experiments

A general and very simple strategy for achieving clean spin-state-selective excitation with full sensitivity in carbon-selective gradient-enhanced 1D HMQC and HSQC pulse schemes is presented. The incorporation of an additional hard 90 degrees (13)C pulse applied along a specific orthogonal axis just prior to acquisition into the conventional sequences allows us to select a simultaneous coherence transfer pathway which usually is not detected. The superimposition of this resulting antiphase magnetization to the conventional in-phase magnetization gives the exclusive excitation of the directly attached proton showing only the alpha or beta spin state of the passive (13)C nucleus. The propagation of this particular spin state to other protons can be accomplished by adding any homonuclear mixing process just after this supplementary pulse. Such an approach affords a suite of powerful selective 1D (13)C-edited NMR experiments which are helpful for resonance assignment purposes in overcrowded proton spin systems and also for the accurate determination of the magnitude and sign of long-range proton-carbon coupling constants in CH spin sytems for samples at natural abundance. Such measurements are performed by measuring the relative displacement of relayed signals in the corresponding alpha and beta 1D subspectra.     

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.     

Flip-back, an old trick to face highly contrasted relaxation times: application in the characterization of pharmaceutical mixtures by CPMAS NMR

The ¹³C–¹H CPMAS with flip-back pulse NMR experiment is revisited in view of applications to pharmaceutical mixtures. The analysis of the kinetics of relaxation and CP transfer with and without the flip-back pulse shows that a significant gain in ¹³C signal can be expected (thus in experimental time) from the flip-back pulse for protons with long T₁. The gain is of the order of T₁ of the protons expressed in seconds. The experiment is applied on samples with highly contrasted spin-lattice relaxation times T₁ for protons, situation encountered in pharmaceutical mixtures. The application of the flip-back increases significantly the relative signal intensity of the component with the longer T₁, making this component detectable even after using short recycle delays. Therefore, this CPMAS with flip-back experiment could be used routinely to get ¹³C CPMAS NMR spectra of mixtures in constant experimental time and signal-to-noise ratio without the need for optimization of the recycle delays, and for whatever may be the degree of crystallinity of the active principal ingredient (API) and/or excipients.     

Assignment of the 1H and 13C Spectra of 5-Methylthio-4H-1-(p-R1-phenyl)-3a-(p-R2-phenyl)-3a,4-dihydro (1,2,4) oxadiazolo(4,5-a), 1,5-benzodiazepines using 2D NMR Spectroscopy.

The ¹H NMR spectra of the title benzodiazepines derivatives is highly congested because all the protons are in aromatic enviroment so many proton signals remain overlap even 300 MHz or higher fields. With this in mind, the assignment of the ¹H and ¹³C spectra of these compounds obtained using COSY, NOESY, HMQC and HMBC experiments is reported.