1H Decoupling Research Articles

Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments.

TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality andhamper data analysis. Specifically, these experiments produce sidebands in the 13C(t 1) dimension with inverted phase corresponding to 1HN resonance frequencies, with approximately 5% intensity of the parent 13C crosspeaks. These artifacts originate from the modulation of the 1HN frequency onto the resonance frequency of 13Cα and/or 13Cβ and are due to 180° pulses imperfections used for 1H decoupling during the 13C(t 1) evolution period. These sidebands can become severe for CAi, CAi-1 and/or CBi, CBi-1 correlation experiments such as TROSY-HNCACB. Here, we implement three alternative decoupling strategies that suppress these artifacts and, depending on the scheme employed, boost the sensitivity up to 14% on Bruker spectrometers. A class of comparable Agilent/Varian pulse sequences that use WALTZ16 1H decoupling can also be improved by this method resulting in up to 60-80% increase in sensitivity.

Solid-State 77Se NMR of Organoselenium Compounds through Cross Polarization Magic Angle Spinning (CPMAS) Method

Characterization of selenium states by 77Se NMR is quite important to provide vital information for mechanism studies in organoselenium-catalyzed reactions. With the development of heterogeneous polymer-supported organoselenium catalysts, the solid state 77Se NMR comes to the spotlight. It is necessary to figure out an advanced protocol that provides good quality spectra within limited time because solid state 77Se NMR measurements are always time consuming due to the long relaxation time and the relatively low sensitivity. Studies on small molecules and several novel polymer-supported organoselenium materials in this article showed that cross polarization (CP) method with the assistance of magic angle spinning (MAS) was more efficient to get high quality spectra than the methods by using single pulse (SP) or high power 1H decoupling (HPHD) combined with MAS. These results lead to a good understanding of the effect of the molecular structure, the heteronuclear coupling, the long-range ordering of the solid (crystal or amorphous), and the symmetry of 77Se on quality of their spectra.

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR.

In DNP MAS NMR experiments at ∼80-110 K, the structurally important -13CH3 and -15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78-300 K. In APG, the three-site hopping of the Ala-Cβ peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C's and 15N's. Similarly, the 15N signal from Ala-NH3+ disappears at ∼173 K, concurrent with the attenuation in CP experiments of other 15N's as well as 13C's. In bR and PI3-SH3, the methyl groups are attenuated at ∼95 K, while all other 13C's remain unaffected. However, both systems exhibit substantial losses of intensity at ∼243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

(17)O NMR Investigation of Water Structure and Dynamics.

The structure and dynamics of the bound water in barium chlorate monohydrate were studied with (17)O nuclear magnetic resonance (NMR) spectroscopy in samples that are stationary and spinning at the magic-angle in magnetic fields ranging from 14.1 to 21.1 T. (17)O NMR parameters of the water were determined, and the effects of torsional oscillations of the water molecule on the (17)O quadrupolar coupling constant (CQ) were delineated with variable temperature MAS NMR. With decreasing temperature and reduction of the librational motion, we observe an increase in the experimentally measured CQ explaining the discrepancy between experiments and predictions from density functional theory. In addition, at low temperatures and in the absence of (1)H decoupling, we observe a well-resolved (1)H-(17)O dipole splitting in the spectra, which provides information on the structure of the H2O molecule. The splitting arises because of the homogeneous nature of the coupling between the two (1)H-(17)O dipoles and the (1)H-(1)H dipole.

High-power 1H composite pulse decoupling provides artifact free exchange-mediated saturation transfer (EST) experiments

Exchange-mediated saturation transfer (EST) provides critical information regarding dynamics of molecules. In typical applications EST is studied by either scanning a wide range of 15N chemical shift offsets where the applied 15N irradiation field strength is on the order of hundreds of Hertz or, scanning a narrow range of 15N chemical shift offsets where the applied 15N irradiation field-strength is on the order of tens of Hertz during the EST period. The 1H decoupling during the EST delay is critical as incomplete decoupling causes broadening of the EST profile, which could possibly result in inaccuracies of the extracted kinetic parameters and transverse relaxation rates. Currently two different 1H decoupling schemes have been employed, intermittently applied 180° pulses and composite-pulse-decoupling (CPD), for situations where a wide range, or narrow range of 15N chemical shift offsets are scanned, respectively. We show that high-power CPD provides artifact free EST experiments, which can be universally implemented regardless of the offset range or irradiation field-strengths.

Comparative studies on manual and automatic backbone chemical shift assignments of 2H/13C/15N-labeled Ube2g1

Ube2g1, one of human E2 enzymes, possesses an additional acidic loop in the vicinity of the active Cys90 residue. The presence of the backbone chemical shifts (bCSs) is essential for various NMR studies of Ube2g1. Triple resonance experiments for the bCSs assignment of 13C/15N-labeled Ube2g1 were not efficient due to the relatively high molecular size (more than 20 kDa). 2H/13C/15N-labeled Ube2g1 was prepared to increase the T2 relaxation time, and then the TROSY-based triple resonance spectra were recorded with 2H decoupling. After the bCSs of Ube2g1 were assigned manually, the resulting bCSs were compared with those from two different automatic assignment programs. The AutoAssign program that utilizes only the peak lists of the NMR spectra accomplished 69 % assignment of the bCSs. On the other hand, the RASPnmr program requires the additional reference CSs that can be predicted from a relevant PDB coordinate. The homologous models of Ube2g1 were generated by various modeling programs, and then their CSs were predicted by using the SHIFTX2 program. The implementations of the predicted bCSs into the RASPnmr analysis resulted in complete and accurate assignment of the bCSs of 2H/13C/15N-labeled Ube2g1. The reference bCSs calculated from various homologous models of Ube2g1 enabled the automatic bCSs assignment process by the RASPnmr program, and a similar application will be possible for the bCS assignments of other proteins.

Aromatic spectral editing techniques for magic-angle-spinning solid-state NMR spectroscopy of uniformly 13C-labeled proteins

The four aromatic amino acids in proteins, namely histidine, phenylalanine, tyrosine, and tryptophan, have strongly overlapping 13C chemical shift ranges between 100 and 160ppm, and have so far been largely neglected in solid-state NMR determination of protein structures. Yet aromatic residues play important roles in biology through π–π and cation–π interactions. To better resolve and assign aromatic residues' 13C signals in magic-angle-spinning (MAS) solid-state NMR spectra, we introduce two spectral editing techniques. The first method uses gated 1H decoupling in a proton-driven spin-diffusion (PDSD) experiment to remove all protonated 13C signals and retain only non-protonated carbon signals in the aromatic region of the 13C spectra. The second technique uses chemical shift filters and 1H–13C dipolar dephasing to selectively detect the Cα, Cβ and CO cross peaks of aromatic residues while suppressing the signals of all aliphatic residues. We demonstrate these two techniques on amino acids, a model peptide, and the microcrystalline protein GB1, and show that they significantly simplify the 2D NMR spectra and both reveal and permit the ready assignment of the aromatic residues' signals.

John Stewart Waugh

John Stewart Waugh, a chemical-physics authority recognized as the founder of the field of high- resolution nuclear magnetic resonance (NMR) in solids, died from complications from Alzheimer’s disease in Lincoln, Massachusetts, on 22 August 2014.John Stewart WaughPPT|High resolutionJohn was born on 25 April 1929 in Storrs, Connecticut, and was raised near the University of Connecticut, where his father, who sparked his interest in science, was a professor of economics and statistics. After high school, John attended Dartmouth College; he graduated in 1949 with highest distinction in chemistry. He then went to graduate school at Caltech, where his thesis supervisor was Donald Yost and his research focused on a topic discovered about three years earlier—NMR. As part of his thesis, “Line profiles in nuclear magnetic resonance absorption,” John assembled a continuous-wave NMR instrument.After receiving his PhD in chemistry and physics in 1953, John returned to the East Coast to be an instructor in the chemistry department at MIT. He was promoted to assistant professor in 1955 and rose through the academic ranks; in 1989 he became an institute professor, the highest honor that MIT bestows on its faculty.During his first 15 years at MIT, John focused his research on various intriguing problems in chemistry and physics that arose during the early years of NMR. For example, he explained the anomalous chemical shifts in 1H NMR spectra of aromatic molecules, coupling in strongly interacting spin systems, and relaxation in liquids and gases. In 1968 he introduced a method for using Fourier-transform spectroscopy to measure spin–lattice relaxation times T1 in complex spin systems. That “inversion recovery” technique remains the method of choice for calculating spin–lattice relaxation rates in gases, liquids, and solids.In 1966 John and engineer Edward Ostroff, who worked at spectrometer manufacturer Magnion, serendipitously discovered that applying a train of intense RF pulses to a spin system in a solid would extend the length of a free induction decay. That led in 1968 to a series of three articles by John, with various coauthors, on multiple-pulse NMR; those papers laid the foundations for high-resolution NMR in solids. One of them, now famous as the WAHUHA experiment, described the initial suppression of the homonuclear dipolar interactions in calcium fluoride and observation of the underlying chemical shifts and their spatial anisotropy, the feature of NMR spectra that renders the technique so useful to physics, chemistry, and biology.Equally important, John and Ulrich Haeberlen introduced a theoretical framework to understand the experiments: the average Hamiltonian theory (AHT), an especially powerful form of time-dependent perturbation theory. Today AHT provides the intellectual underpinnings for spin decoupling, recoupling, and many other stimulating ideas in magnetic resonance and other fields. It is considered an intellectual triumph and the most important theoretical approach in the field.In the early 1970s, John, together with graduate students Alexander Pines and Michael Gibby, published a paper demonstrating that high-resolution NMR experiments could be extended to observe carbon-13, nitrogen-15, phosphorus-31, and other nuclei with smaller dipolar couplings. The experiment’s central feature was the transfer of polarization from abundant spins, namely 1H, to the other nuclei. That circumvented the problematic long T1 of the nuclei. A second feature they incorporated was 1H decoupling, which ensures high resolution. Today that seminal approach to high-resolution NMR is routinely used in labs worldwide.Subsequently, John and his colleagues combined the experiment with multiple-pulse NMR in a manner that reintroduced high-resolution dipole couplings into NMR spectra. That approach allows measurement of internuclear distances. Integration of magic-angle spinning led to what is today known as dipole recoupling, which permits the determination of protein structures in membranes and amyloid fibrils.John is remembered fondly for his well-developed sense of humor. He called the method outlined in his seminal paper on multiple-pulse NMR the WAHUHA experiment after the three authors: Waugh, Huber, and Haeberlen. Following the abandonment of “cycles/second” for the hertz in the early 1970s, John rescued “radian/ second,” the preferred unit in all magnetic resonance calculations, by defining a new unit, the As, so that 1 Hz = 2π As. Aficionados of magnetic resonance use the As to express angular velocity in inverse seconds, or an “Avis.”An avid sailor, John owned many sailboats, including one aptly named Magic Angle and a dinghy called Spin Echo. With Susan, his wife of 31 years, he sailed the coast of Maine, traveled, and raised a succession of beloved Labrador retrievers.John Waugh was a towering figure in NMR and electron paramagnetic resonance, and his intellect, achievements, and wonderful sense of humor were an inspiration to those who knew and worked with him. He will be sorely missed by all of us in the magnetic resonance community.© 2015 American Institute of Physics.

¹³C SPE MAS measurement of ligand concentration in compressible chromatographic beads.

A method for measuring the ligand concentration in heterogeneous materials like chromatography media is described. In this method, 13C single pulse excitation magic angle spinning NMR experiment with broadband 1H decoupling is used to determine the peak integrals for a butyl ligand in the spectrum of a dried chromatography medium. Within a carefully controlled protocol, those integrals compared with that of the internal reference compound dimethyl sulfone provide the required volume concentration with an accuracy of ca 2%. The effects of temperature, degree of hydration, and other experimental parameters are discussed. Copyright © 2015 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.

The Application of Simple and Easy to Implement Decoupling Pulse Scheme Combinations to Effect Decoupling of Large J Values with Reduced Artifacts

Of the various problems in decoupling one nucleus type from another using standard decoupling pulse schemes for broadband decoupling, a particular challenge is to effect full, artifact-free decoupling when the size of the coupling constant is very large. Herein it is demonstrated that 1H decoupling of the 31P NMR spectrum of diethyl phosphonate {HP(=O)(OCH2CH3)2} can be accomplished with reduced artifacts despite the large JH,P1 value of 693 Hz by using a combination of decoupling pulse schemes involving continuous-wave (CW) irradiation and either adiabatic-pulse decoupling (APD), MPF decoupling, or traditional composite-pulse decoupling (CPD) schemes such as WALTZ or GARP. The considered strategy is simple, efficient, and easy to implement on most instruments. The best result was attained for a combination of CW and CPD using GARP with a standard pulse width of 60 μs. Altogether, the advantages of the methodology include low power requirements, complete decoupling, tolerance of a range of large J values, large bandwidth for normal-sized J values, and the suppression of sidebands.

Water scaffolding in collagen: Implications on protein dynamics as revealed by solid‐state NMR

Solid-state NMR studies of collagen samples of various origins confirm that the amplitude of collagen backbone and sidechain motions increases significantly on increasing the water content. This conclusion is supported by the changes observed in three different NMR observables: (i) the linewidth dependence on the 1H decoupling frequency; (ii) 13C CSA changes for the peptide carbonyl groups, and (iii) dephasing rates of 1H-13C dipolar couplings. In particular, a nearly threefold increase in motional amplitudes of the backbone librations about C-Cα or N-Cα bonds was found on increasing the added water content up to 47 wt%D2 O. On the basis of the frequencies of NMR observables involved, the timescale of the protein motions dependent on the added water content is estimated to be of the order of microseconds. This estimate agrees with that from wideline T2(1)H NMR measurements. Also, our wideline 1H NMR measurements revealed that the timescale of the microsecond motions in proteins reduces significantly on increasing the added water content, i.e., an ∼15-fold increase in protein motional frequencies is observed on increasing the added water content to 45 wt% D2 O. The observed changes in collagen dynamics is attributed to the increase in water translational diffusion on increasing the amount of added water, which leads to more frequent "bound water/free water" exchange on the protein surface, accompanied by the breakage and formation of new hydrogen bonds with polar functionalities of protein.

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

Robust and efficient 19F heteronuclear dipolar decoupling using refocused continuous-wave rf irradiation

Refocused continuous wave (rCW) decoupling is presented as an efficient and robust means to obtain well-resolved magic-angle-spinning solid-state NMR spectra of low-γ spins, such as 13C dipolar coupled to fluorine. The rCW decoupling sequences, recently introduced for 1H decoupling, are very robust towards large isotropic and anisotropic shift ranges as often encountered for 19F spins. In rCW decoupling, the so-called refocusing pulses inserted into the CW irradiation eliminate critical residual second- and third-order dipolar coupling and dipolar-coupling against chemical shielding anisotropy cross-terms in the effective Hamiltonian through time-reversal (i.e. refocusing). As important additional assets, the rCW decoupling sequences are robust towards variations in rf amplitudes, operational at low to high spinning speeds, and easy to set-up for optimal performance experimentally. These aspects are demonstrated analytically/numerically and experimentally in comparison to state-of-the-art decoupling sequences such as TPPM, SPINAL-64, and frequency-swept variants of these.

The first observation of Carbon-13 spin noise spectra

We demonstrate the first 13C NMR spin noise spectra obtained without any pulse excitation by direct detection of the randomly fluctuating noise from samples in a cryogenically cooled probe. Noise power spectra were obtained from 13C enriched methanol and glycerol samples at 176MHz without and with 1H decoupling, which increases the sensitivity without introducing radio frequency interference with the weak spin noise. The multiplet amplitude ratios in 1H coupled spectra indicate that, although pure spin noise prevails in these spectra, the influence of absorbed circuit noise is still significant at the high concentrations used. In accordance with the theory heteronuclear Overhauser enhancements are absent from the 1H-decoupled 13C spin noise spectra.

Mechanism of Nanoparticle Formation from Ternary Coground Phenytoin and Its Derivatives

The mechanism of drug nanoparticle formation of phenytoin (DPH) and its derivatives monomethylphenytoin (MDPH) and dimethylphenytoin (DMDPH) was investigated. The drug, polyvinylpyrrolidone K17 (PVP), and sodium dodecyl sulfate were coground to obtain the ground mixture (GM). The DPH GM was amorphous; however, MDPH and DMDPH GMs contained drug crystals. Spectral changes in infrared and (13)C solid-state nuclear magnetic resonance were observed in the DPH GM, partially observed in the MDPH GM, and hardly observed in the DMDPH GM. Mean particle sizes of the DPH, MDPH, and DMDPH GM nanosuspension were almost the same; however, stability after storage differed in the order of DPH > MDPH > DMDPH. The intermolecular interaction between the drug and PVP reflected not only the crystallinity of the drug in the GM but also the stability of the GM suspension. The size and stiffness of drug nanoparticles were evaluated using atomic force microscopy. Crystallization of the amorphous GM and agglomeration of the primary nanocrystals were observed in the DPH GM suspension. In contrast, primary nanocrystals were observed in the DMDPH GM suspension. The size of the drug nanocrystals formed from the different molecular states of the drug in the GM reflects the agglomerated states in water and stability.

NMR Studies of Solid Pentachlorophenol-4-Methylpyridine Complexes Exhibiting Strong OHN Hydrogen Bonds: Geometric H/D Isotope Effects and Hydrogen Bond Coupling Cause Isotopic Polymorphism

We have studied the hydrogen bond interactions of (15)N labeled 4-methylpyridine (4-MP) with pentachlorophenol (PCP) in the solid state and in polar solution using various NMR techniques. Previous spectroscopic, X-ray, and neutron crystallographic studies showed that the triclinic 1:1 complex (4-MPPCP) exhibits the strongest known intermolecular OHN hydrogen bond in the solid state. By contrast, deuteration of the hydrogen bond gives rise to the formation of a monoclinic structure exhibiting a weaker hydrogen bond. By performing NMR experiments at different deuterium fractions and taking advantage of dipolar (1)H-(15)N recoupling under combined fast MAS and (1)H decoupling, we provide an explanation of the origin of the isotopic polymorphism of 4-MPPCP and improve previous chemical shift correlations for OHN hydrogen bonds. Because of anharmonic ground state vibrations, an ODN hydrogen bond in the triclinic form exhibits a shorter oxygen-hydron and a longer oxygen-nitrogen distance as compared to surrounding OHN hydrogen bonds, which also implies a reduction of the local dipole moment. The dipole-dipole interaction between adjacent coupled OHN hydrogen bonds which determines the structure of triclinic 4-MPPCP is then reduced by deuteration, and other interactions become dominant, leading to the monoclinic form. Finally, the observation of stronger OHN hydrogen bonds by (1)H NMR in polar solution as compared to the solid state is discussed.

13C Solid-State NMR of the Mobile Phase of Poly(vinylidene fluoride)

The solid-state 13C spectra of poly(vinylidene fluoride) (PVDF) acquired by direct polarization (DP) with and without simultaneous 19F and 1H decoupling, under moderate sample spinning conditions, ...

Vegetable oil reactions within wood studied by direct 13C excitation with 1H decoupling and magic-angle sample spinning (MAS) NMR

Despite having been used for ages to protect wood against the influence of outdoor elements, the chemistry of vegetable oils within wood is poorly known. We propose a method based on solid-state magic-angle sample spinning NMR to in situ characterize oil oxidation as well as its immobilization. To eliminate signal coming from wood molecules but to keep signal from the oil, direct 13C excitation is performed with low-power 1H decoupling during signal acquisition. To suppress the effect of anisotropic spin-interactions and magnetic field inhomogeneity, the sample is spun at the magic-angle. Mono- and polyunsaturated fatty acid derivatives show a difference in their oxidation process: the monounsaturated methyl oleate reacts with wood components and becomes immobilized while the polyunsaturated methyl linoleate becomes oxidized and form oligomers but does not seem to bind to wood. Linola® oil behaves as would be expected on the basis of its composition by monounsaturated and polyunsaturated chains. This method can be generalized to all coating treatments to characterize chemical pathways and reactions. A better understanding of coating effects on wood is a crucial step to design more efficient protective mixtures.

Dynamic nuclear polarization-enhanced 1H–13C double resonance NMR in static samples below 20 K

We demonstrate the feasibility of one-dimensional and two-dimensional 1H–13C double resonance NMR experiments with dynamic nuclear polarization (DNP) at 9.4T and temperatures below 20K, including both 1H–13C cross-polarization and 1H decoupling, and discuss the effects of polarizing agent type, polarizing agent concentration, temperature, and solvent deuteration. We describe a two-channel low-temperature DNP/NMR probe, capable of carrying the radio-frequency power load required for 1H–13C cross-polarization and high-power proton decoupling. Experiments at 8K and 16K reveal a significant T2 relaxation of 13C, induced by electron spin flips. Carr–Purcell experiments and numerical simulations of Carr–Purcell dephasing curves allow us to determine the effective correlation time of electron flips under our experimental conditions. The dependence of the DNP signal enhancement on electron spin concentration shows a maximum near 80mM. Although no significant difference in the absolute DNP enhancements for triradical (DOTOPA-TEMPO) and biradical (TOTAPOL) dopants was found, the triradical produced greater DNP build-up rates, which are advantageous for DNP experiments. Additionally the feasibility of structural measurements on 13C-labeled biomolecules was demonstrated with a two-dimensional 13C–13C exchange spectrum of selectively 13C-labeled β-amyloid fibrils.

Practical choice of 1H– 1H decoupling schemes in through-bond 1H–{X} HMQC experiments at ultra-fast MAS

Three 1H– 1H homonuclear dipolar decoupling schemes for 1H indirect detection measurements at very fast MAS are compared. The sequences require the following conditions: (i) being operable at very fast MAS, (ii) a long T 2 ′ value, (iii) a large scaling factor, (iv) a small number of adjustable parameters, (v) an acquisition window, (vi) a low rf-power requirement, and (vii) a z-rotation feature. To satisfy these conditions a modified sequence named TIlted Magic-Echo Sandwich with zero degree sandwich pulse (TIMES 0) is introduced. The basic elements of TIMES 0 consist of one sampling window and two phase-ramped irradiations, which realize alternating positive and negative 360° rotations of 1H magnetization around an effective field tilted with an angle θ from the B 0 axis. The TIMES 0 sequence benefits from very large chemical shift scaling factors at ultra-fast MAS that reach κ cs = 0.90 for θ = 25° at ν r = 80 kHz MAS and only four adjustable parameters, resulting in easy setup. Long κ cs T 2 ′ values, where T 2 ′ is a irreversible proton transverse relaxation time, greatly enhance the sensitivity in 1H–{ 13C} through-bond J-HMQC (Heteronuclear Multiple-Quantum Coherence) measurements with 1H– 1H decoupling during magnetization transfer periods. Although similar sensitivity can be obtained with through-space D-HMQC sequences, in which 13C– 1H dipolar interactions are recoupled, J-HMQC experiments incorporating 1H– 1H decoupling benefit from lower t 1-noise, more uniform excitation of both CH, CH 2 and CH 3 moieties, and easier identification of through-bond connectivities.