Simplifying the complex 1H NMR spectra of fluorine-substituted benzamides by spin system filtering and spin-state selection: multiple-quantum-single-quantum correlation

The proton NMR spectra of fluorine-substituted benzamides are very complex (Figure 1) due to severe overlap of (1)H resonances from the two aromatic rings, in addition to several short and long-range scalar couplings experienced by each proton. With no detectable scalar couplings between the inter-ring spins, the (1)H NMR spectra can be construed as an overlap of spectra from two independent phenyl rings. In the present study we demonstrate that it is possible to separate the individual spectrum for each aromatic ring by spin system filtering employing the multiple-quantum-single-quantum correlation methodology. Furthermore, the two spin states of fluorine are utilized to simplify the spectrum corresponding to each phenyl ring by the spin-state selection. The demonstrated technique reduces spectral complexity by a factor of 4, in addition to permitting the determination of long-range couplings of less than 0.2 Hz and the relative signs of heteronuclear couplings. The technique also aids the judicious choice of the spin-selective double-quantum-single-quantum J-resolved experiment to determine the long-range homonuclear couplings of smaller magnitudes.     

J‐Edited Pure Shift NMR for the Facile Measurement of nJHH for Specific Protons

We report a novel 1D J‐edited pure shift NMR experiment (J‐PSHIFT) that was constructed from a pseudo 2D experiment for the direct measurement of proton–proton scalar couplings. The experiment gives homonuclear broad‐band ¹H‐decoupled ¹H NMR spectra, which provide a single peak for chemically distinct protons, and only retain the homonuclear‐scalar‐coupled doublet pattern at the chemical‐shift positions of the protons in the coupled network of a specific proton. This permits the direct and unambiguous measurement of the magnitudes of the couplings. The incorporation of a 1D selective correlation spectroscopy (COSY)/ total correlation spectroscopy (TOCSY) block in lieu of the initial selective pulse, results in the exclusive detection of the correlated spectrum of a specific proton.     

Measurement of long-range 1H-19F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA

Long-range scalar 5J(H1',F) couplings were observed in 5-fluoropyrimidine-substituted RNA. We developed a novel S3E-19F-alpha,beta-edited NOESY experiment for quantitation of these long-range scalar 5J(H1',F) couplings, where the J-couplings can be extracted from inspection of intraresidual (H1',H6) NOE cross-peaks. Quantum chemical calculations were exploited to investigate the relation between scalar couplings and conformations around the glycosidic bond in oligonucleotides. The theoretical dependence of the observed 5J(H1',F) couplings on the torsion angle chi can be described by a generalized Karplus relationship. The corresponding density functional theory (DFT) analysis is outlined. Additional NMR experiments facilitating the resonance assignments of 5-fluoropyrimidine-substituted RNAs are described, and chemical shift changes due to altered shielding in the presence of fluorine-19 (19F) are presented.     

Quenching echo modulations in NMR spectroscopy.

In NMR spectroscopy, homonuclear scalar couplings normally lead to modulations of spin echoes that tend to interfere with the accurate determination of transverse relaxation rates by Carr-Purcell-Meiboom-Gill (CPMG) multiple refocusing experiments. Surprisingly, the echo modulations are largely cancelled when the refocusing pulses applied to the coupling partner deviate slightly from ideal pi rotations due to tilted effective radio-frequency (RF) fields, even at offsets that are much smaller than the radio-frequency amplitude. Experiments and simulations illustrate these effects for two-spin IS systems containing donor and acceptor (15)N nuclei I=N (D) and S=N(A) in RNA Watson-Crick base pairs with homonuclear scalar couplings J(IS)=(2h)J(N(D), N(A)) across the hydrogen bonds.     

Unexpected effects of third-order cross-terms in heteronuclear spin systems under simultaneous radio-frequency irradiation and magic-angle spinning NMR

We recently noted [R. K. Harris, P. Hodgkinson, V. Zorin, J.-N. Dumez, B. Elena, L. Emsley, E. Salager, and R. Stein, Magn. Reson. Chem. 48, S103 (2010)] anomalous shifts in apparent (1)H chemical shifts in experiments using (1)H homonuclear decoupling sequences to acquire high-resolution (1)H NMR spectra for organic solids under magic-angle spinning (MAS). Analogous effects were also observed in numerical simulations of model (13)C,(1)H spin systems under homonuclear decoupling and involving large (13)C,(1)H dipolar couplings. While the heteronuclear coupling is generally assumed to be efficiently suppressed by sample spinning at the magic angle, we show that under conditions typically used in solid-state NMR, there is a significant third-order cross-term from this coupling under the conditions of simultaneous MAS and homonuclear decoupling for spins directly bonded to (1)H. This term, which is of the order of 100 Hz under typical conditions, explains the anomalous behaviour observed on both (1)H and (13)C spins, including the fast dephasing observed in (13)C{(1)H} heteronuclear spin-echo experiments under (1)H homonuclear decoupling. Strategies for minimising the impact of this effect are also discussed.     

Selective internuclear coupling estimation in the solid-state NMR of multiple-spin systems

A new solid-state NMR method is presented for estimating homonuclear dipole-dipole couplings for selected groups of nuclear spins in a multiple-spin coupled network. The methodology combines off-magic-angle spinning, frequency selective spin echoes, and multiple quantum filtering. The new method is insensitive to incoherent relaxation effects and may be used to estimate weak couplings. Internuclear (13)C-(13)C couplings are estimated in uniformly (13)C-labelled l-Histidine·HCl·H(2)O. Weak intermolecular couplings between (13)C nuclei separated by distances exceeding 6 ? are estimated.     

Double quantum 1H MAS NMR studies of hydrogen-bonded protons and water dynamics in materials

Two-dimensional double quantum (DQ) 1H MAS NMR was used to investigate different proton environments in a series of alkali (Na, K, Rb, Cs) [Nb6O19]8- Lindqvist salts, with the water and hydrogen-bound intercluster protons being clearly resolved. Through the analysis of the DQ 1H NMR spinning sideband pattern, it is possible to extract both the mean and distribution of the motionally averaged intramolecular homonuclear 1H-1H dipolar coupling for the different water environments and the intercluster protons. Motional order parameters for the water environments were then calculated from the averaged dipolar couplings. The influence of additional intermolecular dipolar couplings due to multispin interactions were simulated and discussed.     

Theoretical study of the scalar coupling constants across the noncovalent contacts in RNA base pairs: the cis- and trans-watson-crick/sugar edge base pair family

The structure and function of RNA molecules are substantially affected by non-Watson-Crick base pairs actively utilizing the 2'-hydroxyl group of ribose. Here we correlate scalar coupling constants across the noncovalent contacts calculated for the cis- and trans-WC/SE (Watson-Crick/sugar edge) RNA base pairs with the geometry of base to base and sugar to base hydrogen bond(s). 23 RNA base pairs from the 32 investigated were found in RNA crystal structures, and the calculated scalar couplings are therefore experimentally relevant with regard to the binding patterns occurring in this class of RNA base pairs. The intermolecular scalar couplings 1hJ(N,H), 2hJ(N,N), 2hJ(C,H), and 3hJ(C,N) were calculated for the N-H...N and N-H...O=C base to base contacts and various noncovalent links between the sugar hydroxyl and RNA base. Also, the intramolecular 1J(N,H) and 2J(C,H) couplings were calculated for the amino or imino group of RNA base and the ribose 2'-hydroxyl group involved in the noncovalent interactions. The calculated scalar couplings have implications for validation of local geometry, show specificity for the amino and imino groups of RNA base involved in the linkage, and can be used for discrimination between the cis- and trans-WC/SE base pairs. The RNA base pairs within an isosteric subclass of the WC/SE binding patterns can be further sorted according to the scalar couplings calculated across different local noncovalent contacts. The effect of explicit water inserted in the RNA base pairs on the magnitude of the scalar couplings was calculated, and the data for discrimination between the water-inserted and direct RNA base pairs are presented. The calculated NMR data are significant for structural interpretation of the scalar couplings in the noncanonical RNA base pairs.     

High-Resolution Solid-State MAS13C- and 1H-NMR Spectra of Benzenoid Aromatics Adsorbed on Alumina and Silica: Successful Applications of 1D and 2D Pulse Experiments from Liquid-State NMR

Strong line-narrowing effects in solid-state, magic-angle-spinning (MAS) ¹³C- as well as ¹H-NMR spectra of benzenoid aromatics adsorbed at alumina or silica surfAccs indicate high mobility of the organic adsorbates. Even under modest spinning rales (1 kHz), dipolar couplings are sufficiently reduced to allow scalar ^13C,¹H couplings to be measured. Hetero- and homonuclear pulse sequences known from high-resolution NMR in liquids, like SEFT, ^J-RESOLVED, DEPT, COSY, and ¹³C,¹H shift-correlation experiments are successfully applicable. ¹³C spin-lattice relaxation limes are as short as 0.5 s (CH) and 1.1 s (C_q), and T₁(¹H) values are in the order of 0.3 s.     

Determination of 29Si-1H spin-spin coupling constants in organoalkoxysilanes with nontrivial scalar coupling patterns

Application of polarization transfer techniques such as DEPT and INEPT in (29)Si NMR investigation of bridged silane polymerization requires knowledge of indirect (29)Si-(1)H scalar coupling constants in the silane system. However, the fully coupled (29)Si NMR spectra of these molecules, specifically those containing ethylene bridging groups, are too complicated to measure the coupling constants directly by visual inspection. This is because unlike hydrocarbon systems where one-bond proton-carbon coupling constants exceed other coupling constants by an order of magnitude, in silanes the closest proton-silicon pairs are separated by two bonds and all coupling coefficients (both homonuclear and heteronuclear) are of similar magnitude. In these systems, theoretical tools are required to interpret the spectra of even simple molecules. Here, we determine density functional theory estimates of (29)Si-(1)H scalar coupling constants and use these along with homonuclear coupling constant estimates to resolve the nontrivial nature of these spectra. We also report a Karplus equation consistent with the dihedral angle dependence of the three-bond homo- and heteronuclear coupling in the ethylene bridge. By thermal averaging of DFT coupling constants, a good initial guess of the coupled (29)Si spectral pattern is made, which is easily refined by curve fitting to determine estimates of all coupling constants in the system.     

Measurement and Applications of Long‐Range Heteronuclear Scalar Couplings: Recent Experimental and Theoretical Developments

The use of long‐range heteronuclear couplings, in association with ¹H–¹H scalar couplings and NOE restraints, has acquired growing importance for the determination of the relative stereochemistry, and structural and conformational information of organic and biological molecules. However, the routine use of such couplings is hindered by the inherent difficulties in their measurement. Prior to the advancement in experimental techniques, both long‐range homo‐ and heteronuclear scalar couplings were not easily accessible, especially for very large molecules. The development of a large number of multidimensional NMR experimental methodologies has alleviated the complications associated with the measurement of couplings of smaller strengths. Subsequent application of these methods and the utilization of determined J‐couplings for structure calculations have revolutionized this area of research. Problems in organic, inorganic and biophysical chemistry have also been solved by utilizing the short‐ and long‐range heteronuclear couplings. In this minireview, we discuss the advantages and limitations of a number of experimental techniques reported in recent times for the measurement of long‐range heteronuclear couplings and a few selected applications of such couplings. This includes the study of medium‐ to larger‐sized molecules in a variety of applications, especially in the study of hydrogen bonding in biological systems. The utilization of these couplings in conjunction with theoretical calculations to arrive at conclusions on the hyperconjugation, configurational analysis and the effect of the electronegativity of the substituents is also discussed.     

Frequency-selective measurement of heteronuclear scalar couplings in solid-state NMR

A new technique is proposed for selective measurement of heteronuclear scalar J couplings between spins in solids. The method, referred to as FS-J-RES (Frequency-Selective-J-RESolved) NMR, uses frequency-selective irradiation at the I (nonobserved) spin frequency to target a specific pair of spins in a multispin system. In addition, the technique provides direct information about the number of identical I spins chemically bonded to the observed S nucleus. A reference spectrum, recorded without irradiating the I spins, accounts for transverse relaxation, pulse imperfections and dephasing due to homonuclear J couplings between S nuclei, which can be simultaneously measured with this method.     

Observation of CH⋅⋅⋅π Interactions between Methyl and Carbonyl Groups in Proteins

Protein structure and function is dependent on myriad noncovalent interactions. Direct detection and characterization of these weak interactions in large biomolecules, such as proteins, is experimentally challenging. Herein, we report the first observation and measurement of long-range “through-space” scalar couplings between methyl and backbone carbonyl groups in proteins. These J couplings are indicative of the presence of noncovalent C−H⋅⋅⋅π hydrogen-bond-like interactions involving the amide π network. Experimentally detected scalar couplings were corroborated by a natural bond orbital analysis, which revealed the orbital nature of the interaction and the origins of the through-space J couplings. The experimental observation of this type of CH⋅⋅⋅π interaction adds a new dimension to the study of protein structure, function, and dynamics by NMR spectroscopy.     

Observation of CH⋅⋅⋅π Interactions between Methyl and Carbonyl Groups in Proteins

Protein structure and function is dependent on myriad noncovalent interactions. Direct detection and characterization of these weak interactions in large biomolecules, such as proteins, is experimentally challenging. Herein, we report the first observation and measurement of long‐range “through‐space” scalar couplings between methyl and backbone carbonyl groups in proteins. These J couplings are indicative of the presence of noncovalent C−H⋅⋅⋅π hydrogen‐bond‐like interactions involving the amide π network. Experimentally detected scalar couplings were corroborated by a natural bond orbital analysis, which revealed the orbital nature of the interaction and the origins of the through‐space J couplings. The experimental observation of this type of CH⋅⋅⋅π interaction adds a new dimension to the study of protein structure, function, and dynamics by NMR spectroscopy.     

Quantifying millisecond exchange dynamics in proteins by CPMG relaxation dispersion NMR using side-chain 1H probes

A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain (1)H positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {(13)C,(1)H}-glucose and D(2)O a significant number of 'isolated' side-chain (1)H spins are generated, eliminating such effects. It thus becomes possible to record (1)H dispersion profiles at the β positions of Asx, Cys, Ser, His, Phe, Tyr, and Trp as well as the γ positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single (1)H dispersion experiment to 16. The utility of the approach is demonstrated with an application to the four-helix bundle colicin E7 immunity protein, Im7, which folds via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. The extracted (1)H chemical shift differences at side-chain positions provide valuable restraints in structural studies of invisible, excited states, complementing backbone chemical shifts that are available from existing relaxation dispersion experiments.     

Solution state structure determination of silicate oligomers by 29SI NMR spectroscopy and molecular modeling

Evidence for nine new solution state silicate oligomers has been discovered by (29)Si NMR homonuclear correlation experiments of (29)Si-enriched samples. In addition to enhancing signal sensitivity, the isotopic enrichment increases the probability of the (29)Si-(29)Si two-bond scalar couplings that are necessary for the observation of internuclear correlations in 2-D experiments. The proposed assignments are validated by comparisons of experimental and simulated cross-peaks obtained with high digital resolution. The internuclear connectivity indicated by the NMR data suggests that several of these oligomers can have multiple stereoisomers, including conformers and/or diastereomers. The stabilities of these oligomers and their possible stereoisomers have been investigated by electronic structure calculations.     

Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: application to wood lignin

A quantitative method to record (1)H-(13)C correlation NMR spectra (Q-HSQC) is presented. The suppression of (1)J(CH)-dependence is achieved by modulating the polarization transfer delays of HSQC. In addition, the effect of homonuclear couplings, as well as relaxation during the pulse sequence are discussed. We developed the Q-HSQC approach for the quantitative analysis of wood lignin, a complex polymer where it has been difficult to obtain reliable data on the relative amounts of different structural units. The current method is applicable to a variety of complex mixtures, where normal 1D (1)H- and (13)C-NMR methods fail.     

A new NMR method for determining the particle thickness in nanocomposites, using T2,H-selective X{1H} recoupling

A new nuclear magnetic resonance approach for characterizing the thickness of phosphate, silicate, carbonate, and other nanoparticles in organic-inorganic nanocomposites is presented. The particle thickness is probed using the strongly distant-dependent dipolar couplings between the abundant protons in the organic phase and X nuclei (31P, 29Si, 13C, 27Al, 23Na, etc.) in the inorganic phase. This approach requires pulse sequences with heteronuclear dephasing only by the polymer or surface protons that experience strong homonuclear interactions, but not by dispersed OH or water protons in the inorganic phase, which have long transverse relaxation times T2,H. This goal is achieved by heteronuclear recoupling with dephasing by strong homonuclear interactions of protons (HARDSHIP). The pulse sequence alternates heteronuclear recoupling for approximately 0.15 ms with periods of homonuclear dipolar dephasing that are flanked by canceling 90 degrees pulses. The heteronuclear evolution of the long-T2,H protons is refocused within two recoupling periods, so that 1H spin diffusion cannot significantly dephase these coherences. For the short-T2,H protons of a relatively immobile organic matrix, the heteronuclear dephasing rate depends simply on the heteronuclear second moment. Homonuclear interactions do not affect the dephasing, even though no homonuclear decoupling is applied, because long-range 1H-X dipolar couplings approximately commute with short-range 1H-1H couplings, and heteronuclear recoupling periods are relatively short. This is shown in a detailed analysis based on interaction representations. The algorithm for simulating the dephasing data is described. The new method is demonstrated on a clay-polymer nanocomposite, diamond nanocrystals with protonated surfaces, and the bioapatite-collagen nanocomposite in bone, as well as pure clay and hydroxyapatite. The diameters of the nanoparticles in these materials range between 1 and 5 nm. Simulations show that spherical particles of up to 10 nm diameter can be characterized quite easily.     

1H and 13C NMR chemical shift assignments and conformational analysis for the two diastereomers of the vitamin K epoxide reductase inhibitor brodifacoum

Proton and ¹³C NMR chemical shifts and ¹H? ¹H scalar couplings for the two diastereomers of the potent vitamin K epoxide reductase (VKOR) inhibitor brodifacoum have been determined at 293 K from acetone solutions containing both diastereomers. To facilitate difficult assignments, homo‐ and heteronuclear correlation spectra were acquired at 750 and 900 MHz over 268–303 K temperature range. Conformations of both diastereomers inferred from the scalar couplings and 1‐D NOE measurements reveal that one diastereomer (SS/RR) adopts a strained geometry in the cyclohexene ring system of the tetralin group. The NMR spectra also show evidence of line broadening due to conformational exchange at room temperature for the SR/RS diastereomer. These assignments and conformational analyses may be useful in studies of biomolecular interactions of brodifacoum with target proteins such as VKOR and in source determination of brodifacoum. Copyright © 2009 John Wiley & Sons, Ltd.     

A General Method for Extracting Individual Coupling Constants from Crowded 1H NMR Spectra

Couplings between protons, whether scalar or dipolar, provide a wealth of structural information. Unfortunately, the high number of ¹H‐¹H couplings gives rise to complex multiplets and severe overlap in crowded spectra, greatly complicating their measurement. Many different methods exist for disentangling couplings, but none approaches optimum resolution. Here, we present a general new 2D J‐resolved method, PSYCHEDELIC, in which all homonuclear couplings are suppressed in F₂, and only the couplings to chosen spins appear, as simple doublets, in F₁. This approaches the theoretical limit for resolving ¹H‐¹H couplings, with close to natural linewidths and with only chemical shifts in F₂. With the same high sensitivity and spectral purity as the parent PSYCHE pure shift experiment, PSYCHEDELIC offers a robust method for chemists seeking to exploit couplings for structural, conformational, or stereochemical analyses.