Resolution enhancement in spectra of natural products dissolved in weakly orienting media with the help of 1H homonuclear dipolar decoupling during acquisition: application to 1H-13C dipolar couplings measurements

In weakly orienting media such as poly-gamma-benzyl-L-glutamate (PBLG) a polymer that forms a chiral liquid crystal in organic solvents, the spectral resolution for embedded molecules is usually poor because of numerous (1)H, (1)H dipolar couplings that generally broaden proton spectra. Therefore (1)H, (13)C dipolar couplings are difficult or impossible to measure. Here, we incorporate Flip-Flop decoupling during detection into an HSQC experiment. Flip-Flop removes the (1)H, (1)H dipolar couplings and scales the chemical shifts of the protons as well as the (1)H, (13)C dipolar couplings during detection. A resolution gain by a factor 1.5-4.2 and improved signal intensity by an average factor of 1.6-1.7 have been obtained. This technique is demonstrated on (+)-menthol dissolved in a PBLG/CDCl(3) phase.     

Residual 2H quadrupolar couplings in weakly aligned carbohydrates.

We report a novel two-dimensional NMR pulse scheme for the 1H-detected observation of 2H in isotopically 13C, 2H-enriched carbohydrates. This scheme is used for the indirect observation of residual quadrupolar couplings in 13C, 2H-enriched methyl-beta-D-glucopyranoside weakly aligned in a dilute lyotropic liquid-crystalline medium comprising 20% (w/v) dihexanoyl-phosphatidylcholine/dimyristoyl-phosphatidylcholine (1:3 mol/mol) in D2O. The observed residual quadrupolar couplings are substantially larger than residual dipolar one-bond 13C-1H couplings under the same experimental conditions. These quadrupolar couplings are thus a useful alternative to dipolar couplings for the structural analysis of small molecules that align very weakly in dilute liquid-crystalline media. Moreover, since the quadrupolar coupling constant is very uniform throughout endocyclic deuterons of the carbohydrate, these data suggest that adoption of a single average value of this parameter in 2H relaxation studies on the glycan moieties of glycoproteins and glycopeptides is a valid assumption.     

13C-1H HSQC experiment of probe molecules aligned in thermotropic liquid crystals: sensitivity and resolution enhancement in the indirect dimension

The spectra of molecules oriented in liquid crystalline media are dominated by partially averaged dipolar couplings. In the 13C-1H HSQC, due to the inefficient hetero-nuclear dipolar decoupling in the indirect dimension, normally carried out by using a pi pulse, there is a considerable loss of resolution. Furthermore, in such strongly orienting media the 1H-1H and 13C-1H dipolar couplings leads to fast dephasing of transverse magnetization causing inefficient polarization transfer and hence the loss of sensitivity in the indirect dimension. In this study we have carried out 13C-1H HSQC experiment with efficient polarization transfer from 1H to 13C for molecules aligned in liquid crystalline media. The homonuclear dipolar decoupling using FFLG during the INEPT transfer delays and also during evolution period combined with the pi pulse heteronuclear decoupling in the t1 period has been applied. The studies showed a significant reduction in partially averaged dipolar couplings and thereby enhancement in the resolution and sensitivity in the indirect dimension. This has been demonstrated on pyridazine and pyrimidine oriented in the liquid crystal. The two closely resonating carbons in pyrimidine are better resolved in the present study compared to the earlier work [H.S. Vinay Deepak, Anu Joy, N. Suryaprakash, Determination of natural abundance 15N-1H and 13C-1H dipolar couplings of molecules in a strongly orienting media using two-dimensional inverse experiments, Magn. Reson. Chem. 44 (2006) 553-565].     

Anomalous Rotational Resonance Spectra in Magic-Angle Spinning NMR

Magic-angle spinning NMR spectra of samples containing dilute spin-1/2 pairs display broadenings or splittings when a rotational resonance condition is satisfied, meaning that a small integer multiple of the spinning frequency matches the difference in the two isotropic shift frequencies. We show experimental rotational resonance NMR spectra of a 13C2-labeled retinal which are in qualitative disagreement with existing theory. We propose an explanation of these anomalous rotational spectra involving residual heteronuclear couplings between the 13C nuclei and the neighboring 1H nuclei. These couplings strongly influence the rotational resonance 13C spectrum, despite the presence of a strong radiofrequency decoupling field at the 1H Larmor frequency. We model the residual heteronuclear couplings by differential transverse relaxation of the 13C single-quantum coherences. We present a superoperator theory of the phenomenon and describe a numerical algorithm for rapid Liouville space simulations in periodic systems. Good agreement with experimental results is obtained by using a biexponential transverse relaxation model for each spin site.     

Sensitivity-enhanced E.COSY-type HSQC experiments for accurate measurements of one-bond 15N-1H(N) and 15N-13C' and two-bond 13C'-1H(N) residual dipolar couplings in proteins

Novel E.COSY-type HSQC experiments are presented for the accurate measurement of one-bond 15N-1H(N) and 15N-13C(') and two-bond 13C(')-1H(N) residual dipolar couplings in proteins. Compared with existing experiments, the (delta,J)-E.COSY experiments described here are composed of fewer pulses and the resulting spectra exhibit 1.4 times the sensitivity of coupled HSQC spectra. Since residual dipolar couplings play increasingly important roles in structural NMR, the proposed methods should find wide spread application for structure determination of proteins and other biological macromolecules.     

High resolution C nuclear magnetic resonance spectra for symmetrical orthodihalobenzenes including long-range CH couplings

Analysis of high resolution ¹³C NMR spectra for symmetrical orthodihalobenzenes have provided all long-range ¹³CH coupling values in orthodichloro-, dibromo-, and diiodobenzene. Furthermore, since the analyses were sensitive to the sign of the coupling constants, the relative signs of these long-range ¹³CH couplings have been determined from unique spectral fits. The substituent effects on the chemical shifts in this series of compounds appear to be additive. The ¹³CH couplings are compared with coupling values in other compounds and are shown to be related to substituent electronegativity. The absolute magnitude of the ¹³CH couplings for these halogen-substituted compounds are larger than those observed in benzene, with but one exception. In all cases, three-bonded ¹³CH couplings are found to be larger than the two-bond values.     

Interference of homonuclear decoupling and exchange in the solid-state NMR of perfluorocyclohexane

We observe an interference between RF irradiation used for homonuclear decoupling of 19F and conformational exchange in the 13C spectrum of perfluorocyclohexane. We show that these effects can be readily reproduced in simulation, and characterise their dependence on the various NMR and experimental parameters. Their application to observing exchange rates on the kHz timescale is evaluated with respect to T(1rho) measurements and the connections between the two approaches established. The implications for experiments that use homonuclear decoupling of 1H to resolve 1J(CH)couplings in the solid-state are also evaluated in detail.     

A novel liquid crystalline system for partial alignment of polar organic molecules

A new system for partial alignment of polar organic molecules to measure residual dipolar couplings in NMR consists of a 1:1 or a 2:1 mixture of water and DMSO including 3-13% n-alkylpentaethylene glycol as the surfactant. Temperature and concentration dependence of the alignment system are investigated and, as examples, the 13C,1H residual dipolar couplings for the amino acid methionine 1 and for an alpha-methylene-gamma-butyrolactone 2 have been obtained and are compared with those obtained from the alignment media consisting of n-alkylpentaethylene glycol, n-alkyl alcohol and water.     

Long Range 13C Couplings in Hexamethylphosphoramide and Dimethylsulphoxide

One bond ¹³C–¹H couplings have been extensively studied and their magnitudes explained on the basis of two contributing factors, namely the percentage “s” character of the bond¹ and the carbon Z value, or effective nuclear charge². To date however, little has been reported on ¹³C couplings over more than one bond, the main reason being the previous experimental difficulties in measuring these small couplings.     

选择性1NEPT技术及其在FX—90Q仪器上的应用

本文介绍一种~1H—~(13)C远程极化转移的脉冲序列.在JEOL FX—90QNMR波谱仪上实现了这种技术.对一些典型化合物的测定表明,选择性非灵敏核的远程极化转移增强法(SEL1NEPT)在测定季碳原子和进行谱带归属中,是一个有力的工具.     

Measurement of Dipolar Couplings for Methylene and Methyl Sites in Weakly Oriented Macromolecules and Their Use in Structure Determination

A simple and effective method is described for simultaneously measuring dipolar couplings for methine, methylene, and methyl groups in weakly oriented macromolecules. The method is aJ-modulated 3D version of the well-known [¹H-¹³C] CT-HSQC experiment, from which theJand dipolar information are most accurately extracted by using time-domain fitting in the third, constant-time dimension. For CH₂-sites, the method generally yields only the sum of the two individual¹³C-¹H couplings. Structure calculations are carried out by minimizing the deviation between the measured sum, and the sum predicted for each methylene on the basis of the structure. For rapidly spinning methyl groups the dipolar contribution to the splitting of the outer¹³C quartet components can be used directly to constrain the orientation of the C-CH₃bond. Measured sidechain dipolar couplings are in good agreement with an ensemble of NMR structures calculated without use of these couplings.     

Two-dimensional J-resolved and SUPERCOSY NMR experiments in the solid state.

Two 2D experiments, novel to solid-state NMR, are demonstrated using the trimethylsilyl ester of cubic octameric silicate (Q8M8). J-Resolved 13C NMR with BLEW-12 proton homonuclear decoupling reveals the scalar 13C-1H couplings and 29Si SUPERCOSY the connectivities of silicon atoms in the distorted Q8 cube.     

Recoupled polarization-transfer methods for solid-state (1)H--(13)C heteronuclear correlation in the limit of fast MAS

An in-depth account of the effects of homonuclear couplings and multiple heteronuclear couplings is given for a recently published technique for (1)H--(13)C dipolar correlation in solids under very fast MAS, where the heteronuclear dipolar coupling is recoupled by means of REDOR pi-pulse trains. The method bears similarities to well-known solution-state NMR techniques, which form the framework of a heteronuclear multiple-quantum experiment. The so-called recoupled polarization-transfer (REPT) technique is versatile in that rotor-synchronized (1)H--(13)C shift correlation spectra can be recorded. In addition, weak heteronuclear dipolar coupling constants can be extracted by means of spinning sideband analysis in the indirect dimension of the experiment. These sidebands are generated by rotor encoding of the reconversion Hamiltonian. We present generalized variants of the initially described heteronuclear multiple-quantum correlation (HMQC) experiment, which are better suited for certain applications. Using these techniques, measurements on model compounds with (13)C in natural abundance, as well as simulations, confirm the very weak effect of (1)H--(1)H homonuclear couplings on the spectra recorded with spinning frequencies of 25--30 kHz. The effect of remote heteronuclear couplings on the spinning-sideband patterns of CH(n) groups is discussed, and (13)C spectral editing of rigid organic solids is shown to be practicable with these techniques.     

Constraining the anomalous Higgs boson coupling in H+γ production

Higgs boson production in association with a photon(H+) offers a promising channel to test the Higgs boson to photon coupling at various energy scales. Its potential sensitivity to anomalous couplings of the Higgs boson has not been explored with the proton-proton collision data. In this paper, we reinterpret the latest ATLAS H+resonance search results within the Standard Model effective field theory(EFT) framework, using 36.1 fb~(-1) of protonproton collision data recorded with the ATLAS detector at s~(1/2) 13 TeV. Constraints on the Wilson coefficients of dimension-six EFT operators related to the Higgs boson to photon coupling are provided for the first time in the H+final state at the LHC.     

Binuclear spin state selective detection of 1H single quantum transitions using triple quantum coherence: a novel method for enantiomeric discrimination

In the present work a novel methodology is developed for the unambiguous discrimination of enantiomers aligned in chiral liquid crystalline media and the simultaneous determination of 1H-1H and 13C-1H couplings in a single experiment. An INEPT transfer and back transfer of magnetization to protons retain the 13C edited 1H magnetization which is utilized to generate spin selective homonuclear triple quantum coherence of dipolar coupled methyl protons. Spin selective correlation of triple quantum to single quantum coherence results in spin state selective detection by 13C spin and the remaining passive protons. The difference between the successive transitions in the triple quantum dimension pertains to sum of the passive couplings and results in enhanced resolution by a factor of three. This results in unambiguous chiral visualization. The masked 13C satellite transitions in the single quantum spectrum are extracted for chiral discrimination. The technique retains all the passive homo- and heteronuclear couplings in the triple quantum dimension by the application of non-selective refocusing pulse on 1H as well as on 13C spins. This, however, refocuses the chemical shift evolution in the triple quantum dimension, and also overcomes the problem of field inhomogeneity. The method enables the determination of spectral information which is otherwise not possible to derive from the broad and featureless proton spectra. The elegant experimental technique has been demonstrated on different chiral molecules.     

JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules

With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.e., the anti-TROSY component) and thereby make accurate determination of its position difficult. To overcome this problem, a J-evolved transverse relaxation optimized (JE-TROSY) method is presented for the determination of one-bond couplings that involves J-evolution of the sharpest cross-peak multiplet component selected in a TROSY experiment. Couplings are measured from the displacement of the TROSY component in the additional J-evolution dimension relative to a zero frequency origin. The JE-TROSY method is demonstrated on uniformly labeled 15N, 13C-labeled RNA and peptide samples, as well as with an RNA-protein complex, in which the protein is uniformly 15N, 13C-labeled. In all cases, resolved, sensitive spectra are obtained from which heteronuclear one-bond J-couplings could be accurately and easily measured.     

The 2D [31P] spin-echo-difference constant-time [13C, 1H]-HMQC experiment for simultaneous determination of 3J(H3'P) and 3J(C4'P) in 13C-labeled nucleic acids and their protein complexes.

A two-dimensional [31P] spin-echo-difference constant-time [13C, 1H]-HMQC experiment (2D [31P]-sedct-[13C, 1H]-HMQC) is introduced for measurements of 3J(C4'P) and 3J(H3'P) scalar couplings in large 13C-labeled nucleic acids and in DNA-protein complexes. This experiment makes use of the fact that 1H-13C multiple-quantum coherences in macromolecules relax more slowly than the corresponding 13C single-quantum coherences. 3J(C4'P) and 3J(H3'P) are related via Karplus-type functions with the phosphodiester torsion angles beta and epsilon, respectively, and their experimental assessment therefore contributes to further improved quality of NMR solution structures. Data are presented for a uniformly 13C, 15N-labeled 14-base-pair DNA duplex, both free in solution and in a 17-kDa protein-DNA complex.     

Heteronuclear Double-Quantum MAS NMR Spectroscopy in Dipolar Solids

A new pulse sequence for high-resolution solid-state heteronuclear double-quantum MAS NMR spectroscopy of dipolar-coupled spin-12 nuclei is introduced. It is based on the five-pulse sequence known from solution-state NMR, which is here applied synchronously to both spin species. The heteronuclear double-quantum (HeDQ) spinning-sideband patterns produced by this experiment are shown to be sensitive to the heteronuclear distance, as well as the relative orientations of the chemical-shift and dipolar tensors. In particular, it is shown that the HeDQ patterns exhibit an enhanced sensitivity to the chemical shielding tensors as compared with the single-quantum spinning-sideband patterns. The detection of HeDQ patterns via the I and S spins is discussed. The isolated (13)C-(1)H spin pair in deuterated ammonium formate with (13)C in natural abundance was chosen as a model system, and the perturbing influence of dipolar couplings to surrounding protons on the (13)C-(1)H DQ coherence is discussed. The pulse sequence can also be used as a heteronuclear double-quantum filter, hence providing information about heteronuclear couplings, and thus allowing the differentiation of quaternary and CH(n) bonded carbons. The elucidation of (13)C-(1)H dipolar proximities is presented for a sample of bisphenol A polycarbonate with (13)C in natural abundance, recorded with a broadband version of the synchronized five-pulse sequence.     

Proton-detected separated local field spectroscopy

PISEMO, a separated local field experiment that can be performed with either direct (15)N (or (13)C) detection or indirect (1)H detection, is demonstrated on a single crystal of a model peptide. The (1)H signals modulated by (1)H-(15)N heteronuclear dipole-dipole couplings are observed stroboscopically in the windows of the multiple-pulse sequence used to attenuate (1)H-(1)H homonuclear dipole-dipole couplings. (1)H-detection yields spectra with about 2.5 times the signal to noise ratio observed with (15)N-detection under equivalent conditions. Resolution in both the (15)N chemical shift and (1)H-(15)N heteronuclear dipole-dipole coupling dimensions is similar to that observed with PISEMA, however, since only on-resonance pulses are utilized, the bandwidth is better.     

An Efficient NMR Experiment for Analyzing Sugar-Puckering in Unlabeled DNA: Application to the 26-kDa Dead Ringer–DNA Complex

We present a new NMR experiment for estimating the type and degree of sugar-puckering in high-molecular-weight unlabeled DNA molecules. The experiment consists of a NOESY sequence preceded by a constant-time scalar coupling period. Two subexperiments are compared, each differing in the amount of time the (3)J(H3'H2') and (3)J(H3'H2") couplings are active on the H3' magnetization. The resultant data are easy to analyze, since a comparison of the signal intensities of any resolved NOE cross peak originating from H3' atoms of the duplex can be used to estimate the sum of the (3)J(H3'H2') and (3)J(H3'H2") couplings and thus the puckering type of the deoxyribose ring. Isotope filters to eliminate signals of the (13)C-labeled component in the F1-dimension are implemented, facilitating analyses of high-molecular-weight protein-DNA complexes containing (13)C-labeled protein and unlabeled DNA. The utility of the experiment is demonstrated on the 26-kDa Dead Ringer protein-DNA complex and reveals that the DNA uniformly adopts the S-type configuration when bound to protein.