应用相敏HMQC和HSQC谱准确测定异核碳-氢偶合常数

准确测定各种同核和异核偶合常数是核磁共振（NMR）方法研究的一个非常跃的领域。首先,各种三键偶合常数通过Karplus关系式^[1]反映了相应二面角的大小,因此,多键偶合常数的准确测定直接影响分子结构确定的精确性。其次,由于稀液晶溶剂体系NMR方法的发展^[2],准确测定各种异核键偶合常数也显得非常重要,特别是应用场相关偶合常数研究分子在磁场中的取向时,对异核－键偶合常数测定的准确性要求更加严格^[3]。异核－键偶合常数的最准确的测定方法是异核偶合调制的HSQC（Heteronuclear Single-Quantum Coherence)实验^[3],它通过测定一系列异核耦合调制的二维HSQC谱,对交叉峰的强度进行分析来精确确定相应的异核－键偶合常数。这一方法的缺点是比较费时。作者在异核多键偶合常数的准确测定方面也做了一些有意义的工作^[4-6 α]。在前文^[5]工作的基础上,本文提出了二维相敏HMQC（Heteronuclear Multiple-Quantum Coherence)和HSQC（Heteronuclear Single-Quantum Coherence)实验,用于准确测定异核－键偶合常数。     

Using switched angle spinning to simplify NMR spectra of strongly oriented samples

This contribution describes a method that manipulates the alignment director of a liquid crystalline sample to obtain anisotropic magnetic interaction parameters, such as dipolar coupling, in an oriented liquid crystalline sample. By changing the axis of rotation with respect to the applied magnetic field in a spinning liquid crystalline sample, the dipolar couplings present in a normally complex strong coupling spectrum are scaled to a simple weak coupling spectrum. This simplified weak coupling spectrum is then correlated with the isotropic chemical shift in a switched angle spinning (SAS) two-dimensional (2D) experiment. This dipolar-isotropic 2D correlation was also observed for the case where the couplings are scaled to a degree where the spectrum approaches strong coupling. The SAS 2D correlation of C(6)F(5)Cl in the nematic liquid crystal I52 was obtained by first evolving at an angle close to the magic angle (54.7 degrees ) and then directly detecting at the magic angle. The SAS method provides a 2D correlation where the weak coupling pairs are revealed as cross-peaks in the indirect dimension separated by the isotropic chemical shifts in the direct dimension. Additionally, by using a more complex SAS method which involves three changes of the spinning axis, the solidlike spinning sideband patterns were correlated with the isotropic chemical shifts in a 2D experiment. These techniques are expected to enhance the interpretation and assignment of anisotropic magnetic interactions including dipolar couplings for molecules dissolved in oriented liquid crystalline phases.     

Structures of protein-protein complexes are docked using only NMR restraints from residual dipolar coupling and chemical shift perturbations

NMR structures of protein-protein and protein-ligand complexes rely heavily on intermolecular NOEs. Recent work has shown that if no significant conformational changes occur upon complex formation residual dipolar coupling can replace most of the NOE restraints in protein-protein complexes, while restraints derived from chemical shift perturbations can largely replace intermolecular NOEs in protein-ligand structures. By combining restraints from chemical shift perturbations with orientation restraints derived from measurements of residual dipolar couplings, we show that the structure of the EIN-HPr complex can be calculated without NOE restraints. The final structure, built from the crystal structures of EIN and HPr in their uncomplexed form and docked only with NMR restraints, places HPr within 2.5 A of the position determined from the mean NMR structure of the complex.     

On the origin of residual dipolar couplings from denatured proteins

Effects of steric obstruction on random flight chains are examined. Spatial probability distributions are elaborated to calculate residual dipolar couplings and residual chemical shift anisotropy, parameters that are acquired by NMR spectroscopy from solutes dissolved in dilute liquid crystals. Calculations yield chain length and residue position-dependent values in good agreement with simulations to provide understanding of recently acquired data from denatured proteins.     

Solid-state NMR characterization and determination of the orientational order of a nematogen

Thermotropic liquid crystalline compounds are of considerable importance due to their potential applications as advanced functional materials. A mesogen consisting of a terminal dimethylamino group, which can act as a charge-transfer donor, is particularly valuable for its light emission and nonlinear optical properties. In this study, we report the solid-state NMR investigation of the nematic behavior of one such novel mesogen (4-(dodecyloxy)benzoic acid 4-[((4-(dimethylamino)phenyl)imino)methyl]phenyl ester). Static and MAS experiments were performed on nematic and crystalline phases of the compound to measure (13)C chemical shift, (13)C-(1)H dipolar coupling, and (1)H chemical shift values. 2D chemical shift correlation of (1)H and (13)C nuclei confirmed the (13)C chemical shift values determined from 1D CPMAS experiments. The appearance of more peaks in both CPMAS and (13)C-(1)H HETCOR spectra of a crystalline solid suggests the heterogeneous orientations of phenyl rings of the mesogenic core. Variable-temperature experiments infer the motional averaging of these orientations before melting. The (1)H-(13)C dipolar coupling values, measured by 2D PITANSEMA experiments, were used to determine the orientational order of the mesogenic core at various temperatures. The influence of the linking unit and terminal substituents on the order parameter values of the mesogenic core is discussed.     

SESAME-HSQC for simultaneous measurement of NH and CH scalar and residual dipolar couplings

We present a novel pulse sequence, SESAME-HSQC, for the simultaneous measurement of several NH and CH scalar and residual dipolar couplings in double labeled proteins. The proposed Spin-statE Selective All Multiplicity Edited (SESAME)-HSQC combines gradient selected and sensitivity enhanced (15)N- and constant-time (13)C-HSQC experiments with the recently introduced spin-state selective method (Nolis et al., J. Magn. Reson. 180 (2006) 39-50) for measuring couplings simultaneously at amide and aliphatic regions. Excellent resolution and high sensitivity is warranted by removing all coupling interactions during the indirectly detected t(1) period, and by employing pulsed field gradients for coherence selection and utilizing coherence order selective spin-state selection. The scalar and residual dipolar couplings can be readily measured from a two-dimensional (15)N/(13)C-HSQC spectrum without additional spectral crowding. SESAME-HSQC can be used for epitope mapping by observing chemical shift changes in both amide and aliphatic regions. Simultaneously, potential conversion in protein conformation can be probed by analyzing changes in residual dipolar couplings induced by ligand binding. The pulse sequence is experimentally verified with a sample of (15)N/(13)C enriched human ubiquitin. The internuclear vector directions determined from the residual dipolar couplings are found to be in excellent correlation with those predicted from ubiquitin's refined solution structure.     

A 2D solid-state NMR experiment to resolve overlapping aromatic resonances of thiophene-based nematogens

In this study, we demonstrate the feasibility of resolving overlapping 13C chemical shift spectral lines of aromatic rings in a thiophene-based nematogen in the mesophase using a 2D PITANSEMA solid-state NMR method. This technique provided the information about chemical shift values as well as dipolar couplings that are used for determining the orientational order parameter. Large C-H dipolar coupling values measured for thiophene in contrast to phenyl rings suggest that the heterocyclic ring is not part of the molecular axis. Using the order parameter, we determined the orientation of C-H vectors of the thiophene ring. We believe that the 2D solid-state NMR can be extended to other types of liquid crystalline materials such as the banana-based mesogens for determining the orientational order and bent angle.     

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.     

Limits in Proton Nuclear Singlet‐State Lifetimes Measured with para‐Hydrogen‐Induced Polarization

The synthesis of a hyperpolarized molecule was developed, where the polarization and the singlet state were preserved over two controlled chemical steps. Nuclear singlet‐state lifetimes close to 6 min for protons are reported in dimethyl fumarate. Owing to the high symmetry (AA′X₃X₃′ and A₂ systems), the singlet‐state readout requires either a chemical desymmetrization or a long and repeated spin lock. Using DFT calculations and relaxation models, we further determine nuclear spin singlet lifetime limiting factors, which include the intramolecular dipolar coupling mechanism (proton–proton and proton–deuterium), the chemical shift anisotropy mechanism (symmetric and antisymmetric), and the intermolecular dipolar coupling mechanism (to oxygen and deuterium). If the limit of paramagnetic relaxation caused by residual oxygen could be lifted, the intramolecular dipolar coupling to deuterium would become the limiting relaxation mechanism and proton lifetimes upwards of 26 min could become available in the molecules considered here (dimethyl maleate and dimethyl fumarate).     

Measuring amide nitrogen quadrupolar coupling by high-resolution 14N/13C NMR correlation under magic-angle spinning

The measurement of amide nitrogen 14N quadrupolar coupling by two-dimensional 14N/13C correlation experiment is presented with a natural abundant polypeptide. Directly bonded 14N/13C pairs are correlated through J and residual dipolar coupling under magic-angle spinning using a HMQC-type pulse sequence. The 14N quadrupolar coupling is measured from the isotropic second-order quadrupolar shift obtained by comparing the 14N peak positions with the 15N chemical shifts. The high spectral resolution and sensitivity through 13C detection make this method applicable to many organic, inorganic, and biological molecules for the measurement and the use of 14N quadrupolar coupling as a probe for molecular structure and dynamics.     

Determination of sugar structures in solution from residual dipolar coupling constants: methodology and application to methyl beta-D-xylopyranoside

We have developed methodology for the determination of solution structures of small molecules from residual dipolar coupling constants measured in dilute liquid crystals. The power of the new technique is demonstrated by the determination of the structure of methyl beta-d-xylopyranoside (I) in solution. An oriented sample of I was prepared using a mixture of C(12)E(5) and hexanol in D(2)O. Thirty residual dipolar coupling constants, ranging from -6.44 to 4.99 Hz, were measured using intensity-based J-modulated NMR techniques. These include 15 D(HH), 4 (1)D(CH), and 11 (n)D(CH) coupling constants. The accuracy of the dipolar coupling constants is estimated to be < +/- 0.02 Hz. New constant-time HMBC NMR experiments were developed for the measurement of (n)D(CH) coupling constants, the use of which was crucial for the successful structure determination of I, as they allowed us to increase the number of fitted parameters. The structure of I was refined using a model in which the directly bonded interatom distances were fixed at their ab initio values, while 16 geometrical and 5 order parameters were optimized. These included 2 CCC and 6 CCH angles, and 2 CCCC and 6 CCCH dihedral angles. Vibrationally averaged dipolar coupling constants were used during the refinement. The refined solution structure of I is very similar to that obtained by ab initio calculations, with 11 bond and dihedral angles differing by 0.8 degrees or less and the remaining 5 parameters differing by up to 3.3 degrees . Comparison with the neutron diffraction structure showed larger differences attributable to crystal packing effects. Reducing the degree of order by using dilute liquid crystalline media in combination with precise measurement of small residual dipolar coupling constants, as shown here, is a way of overcoming the limitation of strongly orienting liquid crystals associated with the complexity of (1)H NMR spectra for molecules with more than 12 protons.     

Determination of carbon-13 chemical shielding tensor in the liquid state by combining NMR relaxation experiments and quantum chemical calculations

Based on multifield NMR relaxation measurements and quantum chemistry calculations, a strategy aiming at the determination of the chemical shielding tensor (CST) in the liquid state is described. Brownian motions in the liquid state restrict the direct observation of CST to a third of its trace (isotropic shift), and even if CST can be probed indirectly through some spin relaxation rates (specific longitudinal relaxation rates, dipolar chemical shift anisotropy (CSA) cross-correlation rates), an insufficient number of experimental parameters prevents its complete determination. This lack of information can be compensated by using quantum chemical calculations so as to obtain the molecular CST orientation even if a relatively modest level of computation is used. As relaxation parameters involve a dynamic part, a prerequisite is the determination of the molecular anisotropic reorientation which can be obtained independently from dipolar cross-relaxation rates. A polycyclic molecule exhibiting a well-characterized anisotropic reorientation serves as an example for such a study, and some (but not all) carbon-13 chemical shielding tensors can be accurately determined. A comparison with solid-state NMR data and numerous chemical quantum calculations are presented.     

Ion speciation driving chirality transfer in imidazolium-based camphorsulfonate ionic liquid solutions

The underlying principle of the chirality transfer in imidazolium-based camphorsulfonate ionic liquids is rationalized by linking catalytic results from the hydrogenation of [N-(3'-oxobutyl)-N-methylimidazolium] [(+)-camphorsulfonate] to [N-(3'-hydroxybutyl)-N-methylimidazolium] [(+)-camphorsulfonate] in tetrahydrofuran with electrolyte theory by the help of dielectric relaxation spectroscopy. Using this approach we are able to explain why the maximum of the enantiomeric excess of the hydrogenation reaction in tetrahydrofuran is found at a medium concentration of 0.15 mol L(-1), whereas it declines at both, lower and higher concentrations. Dielectric spectra in the concentration range between 0.05 and 1.0 mol L(-1) reveal a solute mode due to dipolar ion pairs and larger dipolar ion clusters. They verify that at very low concentrations the ionic liquid ions are fully solvated with an increasing tendency to form neutral ion pairs with increasing concentration. Already at 0.025 mol L(-1) the degree of dissociation reaches a minimum reflecting a maximum of neutral ion pair formation. With increasing ionic liquid concentration ordered ion clusters are formed by two and more ion pairs. At high concentrations these clusters collapse by dilution in the excess ionic liquid and the defined ion contact necessary for the chirality transfer is lost to a great extent.     

Comparison of aqueous molecular dynamics with NMR relaxation and residual dipolar couplings favors internal motion in a mannose oligosaccharide.

An investigation has been performed to assess how aqueous dynamical simulations of flexible molecules can be compared against NMR data. The methodology compares state-of-the-art NMR data (residual dipolar coupling, NOESY, and (13)C relaxation) to molecular dynamics simulations in water over several nanoseconds. In contrast to many previous applications of residual dipolar coupling in structure investigations of biomolecules, the approach described here uses molecular dynamics simulations to provide a dynamic representation of the molecule. A mannose pentasaccharide, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-D-Manp, was chosen as the model compound for this study. The presence of alpha-linked mannan is common to many glycopeptides, and therefore an understanding of the structure and the dynamics of this molecule is of both chemical and biological importance. This paper sets out to address the following questions. (1) Are the single structures which have been used to interpret residual dipolar couplings a useful representation of this molecule? (2) If dynamic flexibility is included in a representation of the molecule, can relaxation and residual dipolar coupling data then be simultaneously satisfied? (3) Do aqueous molecular dynamics simulations provide a reasonable representation of the dynamics present in the molecule and its interaction with water? In summary, two aqueous molecular dynamics simulations, each of 20 ns, were computed. They were started from two distant conformations and both converged to one flexible ensemble. The measured residual dipolar couplings were in agreement with predictions made by averaging the whole ensemble and from a specific single structure selected from the ensemble. However, the inclusion of internal motion was necessary to rationalize the relaxation data. Therefore, it is proposed that although residual dipolar couplings can be interpreted as a single-structure, this may not be a correct interpretation of molecular conformation in light of other experimental data. Second, the methodology described here shows that the ensembles from aqueous molecular dynamics can be effectively tested against experimental data sets. In the simulation, significant conformational motion was observed at each of the linkages, and no evidence for intramolecular hydrogen bonds at either alpha(1-->2) or alpha(1-->3) linkages was found. This is in contrast to simulations of other linkages, such as beta(1-->4), which are often predicted to maintain intramolecular hydrogen bonds and are coincidentally predicted to have less conformational freedom in solution.     

Dipolar waves as NMR maps of protein structure

The anisotropy of nuclear spin interactions results in a unique mapping of structure to the resonance frequencies and split tings observed in NMR spectra, however, the determination of molecular structure from experimentally measured spectral parameters is complicated by angular ambiguities resulting from the symmetry properties of dipole-dipole and chemical shift interactions. This issue can be addressed through the periodicity inherent in secondary structure elements, which can be used as an index of topology. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels to map protein structure in NMR spectra of both highly and weakly aligned samples. Dipolar waves describe the periodic wavelike variations of the magnitudes of the static heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings (RDCs), in solution NMR spectra. The corresponding properties of the RDCs in solution NMR spectra of weakly aligned helices represent a convergence of solid-state and solution NMR approaches to structure determination.     

Solid-state NMR and quantum chemical investigations of 13Calpha shielding tensor magnitudes and orientations in peptides: determining phi and psi torsion angles

We report the experimental determination of the (13)C(alpha) chemical shift tensors of Ala, Leu, Val, Phe, and Met in a number of polycrystalline peptides with known X-ray or de novo solid-state NMR structures. The 700 Hz dipolar coupling between (13)C(alpha) and its directly bonded (14)N permits extraction of both the magnitude and the orientation of the shielding tensor with respect to the C(alpha)-N bond vector. The chemical shift anisotropy (CSA) is recoupled under magic-angle spinning using the SUPER technique (Liu et al., J. Magn. Reson. 2002, 155, 15-28) to yield quasi-static chemical shift powder patterns. The tensor orientation is extracted from the (13)C-(14)N dipolar modulation of the powder line shapes. The magnitudes and orientations of the experimental (13)C(alpha) chemical shift tensors are found to be in good accord with those predicted from quantum chemical calculations. Using these principal values and orientations, supplemented with previously measured tensor orientations from (13)C-(15)N and (13)C-(1)H dipolar experiments, we are able to predict the (phi, psi, chi(1)) angles of Ala and Val within 5.8 degrees of the crystallographic values. This opens up a route to accurate determination of torsion angles in proteins based on shielding tensor magnitude and orientation information using labeled compounds, as well as the structure elucidation of noncrystalline organic compounds using natural abundance (13)C NMR techniques.     

Investigation of chloromethane complexes of cryptophane‐A analogue with butoxy groups using 13C NMR in the solid state and solution along with single crystal X‐ray diffraction

Host‐guest complexes between cryptophane‐A analogue with butoxy groups (cryptophane‐But) and chloromethanes (chloroform, dichloromethane) were investigated in the solid state by means of magic‐angle spinning ¹³C NMR spectroscopy. The separated local fields method with ¹³C‐¹H dipolar recoupling was used to determine the residual dipolar coupling for the guest molecules encaged in the host cavity. In the case of chloroform guest, the residual dipolar interaction was estimated to be about 19 kHz, consistent with a strongly restricted mobility of the guest in the cavity, while no residual interaction was observed for encaged dichloromethane. In order to rationalize this unexpected result, we performed single crystal X‐ray diffraction studies, which confirmed that both guest molecules indeed were present inside the cryptophane cavity, with a certain level of disorder. To improve the insight in the dynamics, we performed a ¹³C NMR spin‐lattice relaxation study for the dichloromethane guest in solution. The system was characterized by chemical exchange, which was slow on the chemical shift time scale but fast with respect to the relaxation rates. Despite these disadvantageous conditions, we demonstrated that the data could be analyzed and that the results were consistent with an isotropic reorientation of dichloromethane within the cryptophane cavity. Copyright © 2015 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.     

An alternative method for pucker determination in carbohydrates from residual dipolar couplings: a solution NMR study of the fructofuranosyl ring of sucrose

A simple solution NMR method is presented for pucker determination of five-membered rings using only residual dipolar couplings obtained in a single liquid crystalline medium, DMPC/DHPC bicelles (DMPC = dimyristoylphosphatidylcholine; DHPC = dihexanoylphosphatidylcholine). The method was applied to determine the pucker of the fructofuranosyl ring of sucrose. The results indicate a fructofuranosyl pucker phase in the 20 degrees - 70 degrees range. The pucker phases are in agreement with those from previous NMR and optical spectroscopic studies and, importantly, do not rely on empirically parametrized Karplus curves. Furthermore, the analysis implies more than one stable pucker phase and rapid ring interconversion in this range. The present results suggest that using residual dipolar couplings alone can reveal multiple conformations present in solution. Hence, when a sufficient number of residual dipolar coupling constants is measured, the outcome is a robust, reliable, and independent route for determining carbohydrate and nucleic acid structure by NMR spectroscopy.     

Pseudo-CSA restraints for NMR refinement of nucleic acid structure

Upon alignment of oligonucleotides in a magnetic field, the downfield TROSY component of the 13C-{1H} doublet changes its resonance frequency as a result of residual 13C-1H dipolar coupling (RDC) and residual 13C chemical shift anisotropy (RCSA), and the sum of these two second rank tensors is referred to as the pseudo-CSA. The experimentally measured difference in the resonance frequency of the 13C TROSY component in the aligned and isotropic samples is referred to as residual pseudo-CSA (RPCSA), and it can be used directly as a restraint during structure calculation. Because measurement of the RPCSA involves detection of the narrow TROSY 13C doublet component, it is applicable to systems with larger rotational correlation times than RDC measurement. The method is demonstrated for structure refinement of the helical region of a 24-nt stem-loop segment or ribosomal helix-35, uniformly enriched in 13C and 15N, with RPCSA values measured at 5 and 25 degrees C. Substantial cross-validated improvements in structural accuracy are obtained upon incorporation of RPCSA restraints.