Quenching and recoupling of echo modulations in NMR spectroscopy.

Trains of spin echoes are normally modulated by homonuclear scalar couplings. It has long been known that echo modulations are quenched when the pulse-repetition rates are much larger than the offsets of the coupling partners, because the spin systems behave as if they consisted of magnetically equivalent spins when the offsets are suppressed. This type of quenching of the echo modulations can occur when the radio-frequency (RF) pulses are ideal, that is, when they are perfectly homogeneous, properly calibrated to induce rotations through an angle, pi, and have an RF amplitude, omega(1)=-gammaB(1), that is strong compared to the largest offset, Omega(S)=omega(0S)-omega(RF), with respect to the carrier frequency. Recently, it was discovered that echo modulations can also be quenched when the RF pulses are nonideal, that is, when they are too weak to bring about an ideal rotation of the magnetization of the coupling partners, so that the effective fields associated with the RF pulses are tilted in the rotating frame. This phenomenon typically occurs when the pulse-repetition rates are much slower than the offset of the coupling partner. Under such conditions, it turns out, however, that for certain offsets, when the phase, Phi(S) (which arises from a free precession of the magnetization of the coupling partner, S, in the pulse interval, 2tau, and the pulse length, tau(pi)), approaches a multiple of 2pi, the echo modulations are restored. However, the frequencies of these echo modulations are not simply determined by the homonuclear scalar coupling, J(IS). The Fourier transforms of the echo trains (the so-called "J spectra") reveal surprising multiplet patterns, and the amplitudes of the echo modulations depend on the offsets of the coupling partners. Herein, we present a unified theory, based on an average-Hamiltonian approach, to describe these effects for two-spin systems. Experimental evidence of echo modulations in a system of two spins is presented. Experiments with three and more spins, backed up by extensive numerical simulations, will be presented elsewhere.     

Low-power composite CPMG HSQMBC experiment for accurate measurement of long-range heteronuclear coupling constants

A modified version of CPMG-HSQMBC pulse scheme is presented for the measurement of long-range heteronuclear coupling constants. The method implements adiabatic inversion and refocusing pulses on the heteronucleus. Low-power composite 180° XY-16 CPMG pulse train is applied on both proton and X nuclei during the evolution of long-range couplings to eliminate phase distortions due to co-evolution of homonuclear proton-proton couplings. The pulse sequence yields pure absorption antiphase multiplets allowing precise and direct measurement of the (n)J(XH) coupling constants regardless from the size of the proton-proton couplings. The applicability of the method is demonstrated using strychnine as a model compound. The selective 1D version of the method is also presented.     

Determination of (1)H homonuclear scalar couplings in unlabeled carbohydrates

The scarcity of structural information on carbohydrates results from combined difficulties to crystallize and the limitations in NMR analysis. Current methods for determining basic NMR parameters such as (1)H homonuclear scalar couplings are very limited, especially for large molecules such as polysaccharides, oligonucleotides, and the carbohydrate part of glycoproteins. In this paper, a NMR experiment for the determination of endocyclic (1)H homonuclear scalar couplings ((3)J(HH)) in unlabeled carbohydrates is presented. In addition to scalar couplings, cross-correlated dipole-dipole relaxation rates were measured for large polysaccharides. The measurement of all endocyclic homonuclear coupling constants within monosaccharide units is presented for lactose, a model disaccharide, and for a natural-abundance 2 MDa bacterial polysaccharide excreted by Streptococcus thermophilus Sfi39.     

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.     

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.     

Frequency-selective homonuclear dipolar recoupling in solid state NMR

We introduce a new approach to frequency-selective homonuclear dipolar recoupling in solid state nuclear magnetic resonance (NMR) with magic-angle spinning (MAS). This approach, to which we give the acronym SEASHORE, employs alternating periods of double-quantum recoupling and chemical shift evolution to produce phase modulations of the recoupled dipole-dipole interactions that average out undesired couplings, leaving only dipole-dipole couplings between nuclear spins with a selected pair of NMR frequencies. In principle, SEASHORE is applicable to systems with arbitrary coupling strengths and arbitrary sets of NMR frequencies. Arbitrary MAS frequencies are also possible, subject only to restrictions imposed by the pulse sequence chosen for double-quantum recoupling. We demonstrate the efficacy of SEASHORE in experimental (13)C NMR measurements of frequency-selective polarization transfer in uniformly (15)N, (13)C-labeled L-valine powder and frequency-selective intermolecular polarization transfer in amyloid fibrils formed by a synthetic decapeptide containing uniformly (15)N, (13)C-labeled residues.     

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.     

Characterization of the cation and temperature dependence of DNA quadruplex hydrogen bond properties using high-resolution NMR

Variations in the hydrogen bond network of the Oxy-1.5 DNA guanine quadruplex have been monitored by trans-H-bond scalar couplings, (h2)J(N2N7), for Na(+)-, K(+)-, and NH(4)(+)-bound forms over a temperature range from 5 to 55 degrees C. The variations in (h2)J(N2N7) couplings exhibit an overall trend of Na(+) > K(+) > NH(4)(+) and correlate with the different cation positions and N2-H2...N7 H-bond lengths in the respective structures. A global weakening of the (h2)J(N2N7) couplings with increasing temperature for the three DNA quadruplex species is accompanied by a global increase of the acceptor (15)N7 chemical shifts. Above 35 degrees C, spectral heterogeneity indicates thermal denaturation for the Na(+)-bound form, whereas spectral homogeneity persists up to 55 degrees C for the K(+)- and NH(4)(+)-coordinated forms. The average relative change of the (h2)J(N2N7) couplings amounts to approximately 0.8 x 10(-3)/K and is thus considerably smaller than respective values reported for nucleic acid duplexes. The significantly higher thermal stability of H-bond geometries in the DNA quadruplexes can be rationalized by their cation coordination of the G-quartets and the extensive H-bond network between the four strands. A detailed analysis of individual (h2)J(N2N7) couplings reveals that the 5' strand end, comprising base pairs G1-G9* and G4*-G1, is the most thermolabile region of the DNA quadruplex in all three cation-bound forms.     

Measurement of long-range cross-correlation rates using a combination of single- and multiple-quantum NMR spectroscopy in one experiment

A method is described to determine long-range cross-correlations between the modulations of an anisotropic chemical shift (e.g., of a C' carbonyl carbon in a protein) and the fluctuations of a weak long-range dipolar interaction (e.g., in cross-correlation between the same C' carbonyl and the H(N) proton of the neighboring amide group). Such long-range correlations are difficult to measure because the corresponding long-range scalar couplings are so small that Redfield's secular approximation is often violated. The method, which combines features of single- and double-quantum NMR spectroscopy, allows one to cancel the effects of dominant short-range dipolar interactions (e.g., between the CSA of the amide nitrogen N and the dipolar coupling to its attached proton H(N)) and is designed so that the secular approximation is rescued even if the scalar coupling between the long-range dipolar coupling partners is very small. The cross-correlation rates thus determined in ubiquitin cover a wide range because of local motions and variations of the CSA tensors.     

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.     

Spin selective multiple quantum NMR for spectral simplification, determination of relative signs, and magnitudes of scalar couplings by spin state selection

In the present work we demonstrate a novel method for spectral simplification and determination of the relative signs of the scalar couplings using a spin selective multiple quantum NMR experiment. A spin selective excitation of double quantum coherence of A and M spins in a weakly coupled three spin system of the type AMX, results in a doublet in the double quantum dimension whose separation corresponds to the sum of couplings of the active spins to the passive spin X. One component of the doublet has the passive spin X in mid R:alpha state while the other component has the passive spin X in mid R:beta state. The spin selective conversion of double quantum coherence to single quantum coherence does not disturb the spin states of the passive spin thereby providing the spin state selection. There will be two domains of single quantum transitions in single quantum dimension at the chemical shift positions of A and M spins. The mid R:alpha domain of A spin is a doublet because of mid R:alpha and mid R:beta states of M spin only, while that of mid R:beta domain is another doublet in a different cross section of the spectra. The scalar coupling J(AM) can be extracted from any of the mid R:alpha and mid R:beta domain transitions while the relative displacements of the two doublets between the two domains at the two chemical shifts provides the magnitude and sign of the scalar coupling J(AX) relative to the coupling J(MX). Similar result is obtained for zero quantum studies on AMX spin system. The proposed technique is discussed theoretically using product operator approach. The new spin state selective double quantum J-resolved sequence has also been developed. The methodology is confirmed experimentally on a homonuclear weakly coupled three spin system and applied to two different heteronuclear five spin systems.     

Scalar Coupling between the 15N Centres in Methylated 1,8‐Diaminonaphthalenes and 1,6‐Diazacyclodecane: To What Extent is 2HJNN a Reliable Indicator of N?N Distance?

The scalar couplings between hydrogen bonded nitrogen centres ((2H)J(NN)) in the free-base and protonated forms of the complete series of [(15)N(2)]-N-methylated 1,8-diamino naphthalenes in [D(7)]DMF solution have been determined, either directly (15N[1H] NMR), or, indirectly (13C[1H] NMR and simulation of the X part of the ABX spectrum (X=13C, A,B=15N)). Additionally, the (2H)J(NN) value in the HBF(4) salt of [(15)N(2)]-1,6-dimethyl-1,6-diazacyclodecane was determined, indirectly by 13C[(1H] NMR spectroscopy. As confirmed by DFT calculations and by reference to CSD, the rigid nature of the naphthalene scaffold results in rather low deviations in N,N distance or H-N,N angle within each series, apart from the free base of the permethylated compound (proton sponge) where the naphthalene ring is severely distorted to relieve strain. Despite such restrictions, the (2H)J(NN) values increase smoothly from 1.5 to 8.5 Hz in the protonated series as the degree of methylation increases. The effect in the free-base forms is much less pronounced (2.9 to 3.7 Hz) with no scalar N,N coupling detected in the permethylated compound (proton sponge) due to the lack of hydrogen bond between the N,N centres. Neither the pK(a) nor the N-N distance in the protonated forms correlates with (2H)J(NN). However, the sum of the (13)C NMR shifts of the naphthalene ring C(1,8) carbons which are attached directly to the nitrogen centres correlates linearly with (2H)J(NN) and with the degree of methylation. The gas-phase computed (2H)J(NN) is almost constant throughout the homologous series, and close to the experimental value for the tetramethylated ion. However, the computed coupling constant is attenuated in structures involving microsolvation of each N-H unit, and the trend then matches experiment. These experimental and computational observations suggest that Fermi contact between the two N centres is decreased upon formation of strong charge-dispersing intermolecular hydrogen bonds of the free N-H groups with the solvent.     

3D TEDOR NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly (13)C,(15)N-labeled solids

We describe three-dimensional magic-angle-spinning NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly (13)C,(15)N-labeled solids. The approaches employ transferred echo double resonance (TEDOR) for (13)C-(15)N coherence transfer and (15)N and (13)C frequency labeling for site-specific resolution, and build on several previous 3D TEDOR techniques. The novel feature of the 3D TEDOR pulse sequences presented here is that they are specifically designed to circumvent the detrimental effects of homonuclear (13)C-(13)C J-couplings on the measurement of weak (13)C-(15)N dipolar couplings. In particular, homonuclear J-couplings lead to two undesirable effects: (i) they generate anti-phase and multiple-quantum (MQ) spin coherences, which lead to spurious cross-peaks and phase-twisted lines in the 2D (15)N-(13)C correlation spectra, and thus degrade the spectral resolution and prohibit the extraction of reliable cross-peak intensities, and (ii) they significantly reduce cross-peak intensities for strongly J-coupled (13)C sites (e.g., CO and C(alpha)). The first experiment employs z-filter periods to suppress the anti-phase and MQ coherences and generates 2D spectra with purely absorptive peaks for all TEDOR mixing times. The second approach uses band-selective (13)C pulses to refocus J-couplings between (13)C spins within the selective pulse bandwidth and (13)C spins outside the bandwidth. The internuclear distances are extracted by using a simple analytical model, which accounts explicitly for multiple spin-spin couplings contributing to cross-peak buildup. The experiments are demonstrated in two U-(13)C,(15)N-labeled peptides, N-acetyl-L-Val-L-Leu (N-ac-VL) and N-formyl-L-Met-L-Leu-L-Phe (N-f-MLF), where 20 and 26 (13)C-(15)N distances up to approximately 5-6 A were measured, respectively. Of the measured distances, 10 in N-ac-VL and 13 in N-f-MLF are greater than 3 A and provide valuable structural constraints.     

Triple oscillating field technique for accurate distance measurements by solid-state NMR

We present a new concept for homonuclear dipolar recoupling in magic-angle-spinning (MAS) solid-state NMR experiments which avoids the problem of dipolar truncation. This is accomplished through the introduction of a new NMR pulse sequence design principle: the triple oscillating field technique. We demonstrate this technique as an efficient means to accomplish broadband dipolar recoupling of homonuclear spins, while decoupling heteronuclear dipolar couplings and anisotropic chemicals shifts and retaining influence from isotropic chemical shifts. In this manner, it is possible to synthesize Ising interaction (2IzSz) Hamiltonians in homonuclear spin networks and thereby avoid dipolar truncation--a serious problem essentially all previous homonuclear dipolar recoupling experiments suffer from. Combination of this recoupling concept with rotor assisted dipolar refocusing enables easy readout of internuclear distances through comparison with analytical Fresnel curves. This forms the basis for a new class of solid-state NMR experiments with potential for structure analysis of uniformly 13C labeled proteins through accurate measurement of 13C-13C internuclear distances. The concept is demonstrated experimentally by measurement of C alpha-C', C beta-C', and C gamma-C' internuclear distances in powder samples of the amino acids L-alanine and L-threonine.     

Simplification of the 1H NMR spectra of enantiomers dissolved in chiral liquid crystals, combining variable angle sample spinning and selective refocusing experiments

This work presents a technique to simplify overcrowded proton spectra in chiral liquid crystal solvents using rotation of the sample near the magic angle, VASS, combined with homonuclear selective refocusing 2D NMR experiments, SERF. This methodology provides a powerful tool to visualise enantiomers out of unresolved proton spectra. A modified SERF sequence is presented where the resulting 2D spectrum can be phased to increase the resolution. Accurate enantiomeric excesses are determined that are not possible to measure on static samples. Two examples are presented.     

Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme.

A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.     

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.     

NMR measurements of scalar-coupling distributions in disordered solids

The measurement of scalar (J) couplings by solid-state NMR is a field of great interest, since this interaction is a rich source of local structural information, complementary to dipolar and chemical shift interactions. Here, we first demonstrate that J-coupling distributions exist and can be observed in disordered solids, as illustrated with the observation of a pair-specific distribution of (2)J((31)P-N-(31)P) couplings in a bis-phosphino amine, and we investigate the potential effects of such distributions on the measurement of average J-coupling constants. Second, we show that the measurement of two-dimensional (2D) distributions of J-couplings provides a much richer probe of local structural disorder than one-dimensional distributions, and we introduce new methods that provide different (selective or non-selective) ways of measuring 2D J distributions in a wide range of disordered systems. These methods are finally applied to a slightly disordered polymorphic sample of fully (13)C-enriched cellulose, and then to the bis-phosphino amine sample, from which 2D (2)J(PP)-coupling distributions are clearly identified and interpreted.     

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.     

Spectra edited by relative signs of homonuclear couplings of low abundance nuclei

The proposed homonuclear coupling sign edited (HCSE) experiment can detect signed homonuclear couplings between low abundant nuclei like ¹³C, ²⁹Si and ¹⁵N in linear spin systems, that is, in systems where two nuclei are coupled by the measured coupling, and one of them is coupled by a second coupling to a nucleus of different kind. The third nucleus is usually high abundant hydrogen. Two spectra are measured during the HCSE experiment. Their weighed sum and difference yield two other spectra, one containing peaks coupled only by positive measured couplings and the other having peaks coupled by negative measured couplings. The usual E.COSY‐type experiment requires all three couplings in the three spin system (triangular spin system) and not only two couplings as the HCSE experiment. The experiment was successfully tested on known carbon–carbon and silicon–silicon two bond couplings. A set of six simple siloxanes with |²J(Si‐O‐Si)| couplings ranging from 0.5 to 9.0 Hz was measured for the first time, and all the couplings were found to be positive. Copyright © 2012 John Wiley & Sons, Ltd.