Optimizing delays in the MBOB,broadband HMBC,and broadband XLOC NMR pulse sequences

The MBOB, broadband HMBC, and broadband XLOC NMR pulse sequences (A. Meissner and O. W. S?rensen (2000, Magn. Reson. Chem. 38, 981-984; 2001, 39, 49-52)) were introduced as a means of obtaining heteronuclear long-range correlation spectra with broadband excitation over an interval of heteronuclear long-range J coupling constants. However, it is not trivial what combination of delays to choose for a given purpose, particularly if one-bond and long-range correlation spectra are obtained simultaneously as in MBOB. This paper presents a way to determine sets of delays for MBOB, broadband HMBC, and broadband XLOC resolving the problem. The results tabulated suit various ranges of J coupling constants and transverse relaxation times.     

Accurate determination of small one-bond heteronuclear residual dipolar couplings by F1 coupled HSQC modified with a G-BIRD(r) module

We report a G-BIRD(r) modified coupled HSQC experiment for the accurate determination of one-bond heteronuclear residual dipolar couplings. The G-BIRD(r) module has been employed to refocus the long-range coupling evolution of the heteronucleus during the t1 frequency labeling period. As a result, the crosspeaks obtained are split by only the direct one-bond coupling that can be extracted by measuring simple frequency differences between singlet maxima. Additionally the decoupling of long-range multiple bond splittings leads to considerable sensitivity enhancement. The modification also has been applied in a TROSY sequence resulting in a significant sensitivity and resolution improvement.     

J-modulated ADEQUATE (JM-ADEQUATE) experiment for accurate measurement of carbon-carbon coupling constants

A new method for the accurate determination of carbon-carbon coupling constants is described. The method is based on a modified ADEQUATE experiment, where a J-modulated spin-echo sequence precedes the ADEQUATE pulse scheme. The J-modulation and scaling of carbon-carbon couplings is based on simultaneous incrementation of 13C chemical shift and coupling evolution periods. The time increment for the homonuclear carbon-carbon coupling evolution can be suitably scaled with respect to the corresponding increment for the chemical shift evolution. Typically a scaling factor of 2 to 3 is employed for the measurement of one-bond coupling constants, while multiplication by a factor of 10 to 15 is applied when small long-range couplings are determined. The same pulse scheme with coupling evolution period optimized for one-bond or long-range couplings allows the determination of the corresponding carbon-carbon coupling constants. The splittings of the ADEQUATE crosspeaks in the F1 dimension yield the appropriately multiplied coupling constants.     

Sensitivity improvement and new acquisition scheme of heteronuclear active-coupling-pattern-tilting spectroscopy

A simplified phase-cycling scheme for heteronuclear active-coupling-pattern tilting (ACT) spectroscopy is presented. It is demonstrated that the theoretically expected twofold sensitivity gain over earlier implementations can be experimentally realized. A further intensity increase by a factor of 2 is obtained with standard sensitivity-enhancement pulse-sequence elements. The HSQC-HECADE sequence presented is designed for an accurate determination of heteronuclear one-bond and, with subsequent I-spin isotropic mixing, long-range coupling constants. As an exemplary application, the determination of the (3)J(N,Hbeta) coupling constants in a peptide at natural isotope abundance is demonstrated. Additionally, a new polarization-transfer step for the long-range HSQC-HECADE experiment is proposed which avoids a fixed delay tuned to a specific coupling-constant value. Thus, the long-range correlation experiment does not require prior knowledge of the coupling constants to be measured and yields more uniform cross-peak intensity for a broad range of active coupling constants.     

Experiments to detect long-range heteronuclear shift correlations: LR-J-HSMQC

The utility of the J-HSMQC experiment to detect long-range CH correlations was investigated. Two new long-range J-compensated pulse sequences, LR-J-HSMQC(80,27) and LR-J-HSMQC(27,80), were developed using the (3beta(x))beta(y) composite 90 degrees pulse sequence. These two experiments were shown to be effective for long-range coupling constants, (n)J(CH), that were greater than 3 Hz. Although the overall sensitivities of the long-range J-HSMQC experiments were slightly lower than that of the conventional decoupled HMBC experiment, their 2D maps showed additional cross peaks that could be useful in structure elucidation. LR-J-HSMQC(27,80) was very efficient in yielding two- and four-bond relay correlations. The utility of the new sequences is demonstrated with strychnine as the sample.     

HMSC: simultaneously detected heteronuclear shift correlation through multiple and single bonds

A new 2D pulse sequence HMSC (heteronuclear multiple-bond and single-bond coupling connectivities) for the simultaneous detection of long-range and one-bond heteronuclear connectivities is proposed which allows the two types of responses to be separated and the corresponding (n)J(CH) and (1)J(CH) connectivity maps to be calculated. (n)J(CH) coherences are selectively labeled in the course of the pulse sequence, the correspondingly acquired data are separately stored, and a simple add/subtract procedure is applied to disentangle and edit (n)J(CH) and (1)J(CH) responses prior to final data processing. Unlike standard methods, which are designed to measure one single type of heteronuclear spin-spin interactions and to efficiently suppress the other, both (n)J(CH) and (1)J(CH) are measured simultaneously in a single experiment with the HMSC pulse sequence. Compared to the common strategy with two standard experiments applied one after the other, e.g., HMBC and HMQC, valuable measuring time may be saved with this single experiment approach. The efficiency of the new pulse sequence and the quality of the corresponding spectra are demonstrated using strychnine. Features such as sensitivity, lineshapes, and the suppression of (1)J(CH) residual peaks in the final (n)J(CH) subspectra are investigated and compared with the corresponding results obtained with standard methods. The attractive and unique single experiment approach, its high efficiency, and its easy experimental setup together with straightforward data processing make HMSC a valuable experimental alternative for the today's more time-consuming "two-step" practice and makes it suitable for standard routine applications.     

Accurate and Precise Measurement of Heteronuclear Long-Range Couplings by a Gradient-Enhanced Two-Dimensional Multiple-Bond Correlation Experiment

We propose a phase-sensitive gradient-enhanced two-dimensional heteronuclear multiple-bond correlation (psge-2D HMBC) experiment for speedy, accurate, and precise measurement of²J_CHand³J_CH. The experiment does not suppress one-bond correlations. Rather, the value of a desired long-rangeJ_CHis obtained from the pertinent cross-peak pattern in the HMBC spectrum, using the corresponding¹J_CHcorrelation pattern as reference. The application of the proposed experiment is illustrated for the trisaccharide raffinose.     

The CLIP/CLAP-HSQC: pure absorptive spectra for the measurement of one-bond couplings

Heteronuclear residual dipolar one-bond couplings of organic molecules at natural abundance are most easily measured using t2 coupled HSQC spectra. However, inevitably mismatched transfer delays result in phase distortions due to residual dispersive antiphase coherences in such experiments. In this article, slightly modified t2 coupled HSQC experiments with clean inphase (CLIP) multiplets are introduced which also reduce the intensities of undesired long-range cross peaks. With the corresponding antiphase (CLAP) experiment, situations where alpha and beta components overlap can be resolved for all multiplicities in an IPAP manner. A comparison of the experiments using hard pulses and shaped broadband excitation and inversion pulses on the heteronucleus is given and potential spectral artefacts are discussed in detail.     

Accurate measurement of small heteronuclear coupling constants from pure-phase α/β HSQMBC cross-peaks

A simple proton-selective α/β-HSQMBC experiment is proposed for the accurate measurement of long-range proton-carbon coupling constants (nJCH) in small molecules without need for an individualized and time-consuming post-processing fitting procedure. The method acquires two pure-phase In-phase (IP) and Anti-phase (AP) multiplets completely free of any phase distortion due to the absence of JHH evolution. Accurate nJCH values can be directly measured analyzing the relative displacement of the resulting IPAP cross-peaks. Discussion about signal intensity dependence and cross-talk is made for a range of experimental conditions. The robustness of the method is evaluated by comparing the nJCH value measured from the analysis of the three available IP, AP and IPAP multiplet patterns. Multiple-frequency and region-selective versions of the method can also be efficiently recorded provided that excited protons are not mutually coupled.     

Getting to know the nitrogen next door: HNMBC measurements of amino sugars

Long-range 1H-1?N correlations detected by the heteronuclear multiple-bond correlation (HMBC) experiment are explored for the characterization of amino sugars. The gradient-enhanced HMBC, IMPACT-HMBC, and a modified pulse sequence with the 1J-filters removed, IMPACT-HNMBC, are compared for sensitivity and resolution. 1?N chemical shifts and long-range proton correlations are reported using the IMPACT-HNMBC experiment for N-acetyl-glucosamine, N-acetyl-galactosamine, and for a series of glucosamine analogs with an N-sulfo substitution, unmodified amino group, and 6-O-sulfonation. As is common with sugars, for all the compounds examined both anomeric forms are present in solution. For each compound studied, the 1?N chemical shifts of the α anomer are downfield of the β form. For the N-acetylated sugars, the β anomer has a unique long-range 1?N correlation to the anomeric proton not observed for the α anomer. Though N-sulfonation results in a significant change in the 1?N chemical shift of the glucosamine analogs, 6-O sulfo substitution has no significant effect on the local environment of the amino nitrogen. For N-acetylated sugars in D?O solution, peaks in the 1?N projection of the HMBC spectrum appear as triplets as a result of J-modulation due to 2H-1?N coupling.     

HAT HMBC: a hybrid of H2BC and HMBC overcoming shortcomings of both

A new 2D NMR experiment, HAT HMBC, that is a hybrid of H2BC and HMBC aims at establishing two-bond correlations absent in H2BC spectra because of vanishing (3)J(HH) coupling constants. The basic idea is to create an additional pi phase difference in the multiplet structure in HMBC peaks with respect to the (n+1)J(HH) coupling constant between the proton(s) attached to a (13)C and a (1)H separated by n bonds. Thus HMBC peaks associated with small J(HH) will be the most attenuated in a HAT HMBC spectrum in comparison to a regular HMBC spectrum, i.e. peaks associated with (n+1)J(HH) and (n)J(CH) will for n>2 usually be strongly attenuated. The HAT HMBC pulse sequences contain the same number of pulses as regular HMBC and are only a few milliseconds longer.     

Band-Selective HSQC and HMBC Experiments Using Excitation Sculpting and PFGSE

Variants of the HSQC and HMBC experiments are described. They allow the restriction of the heteronuclear chemical shift domain without causing spectral folding. Selectivity is introduced in the HSQC experiment by means of excitation sculpting. The selective element of the pulse sequence is a double pulsed field gradient spin echo. It may be used either split by the t(1) evolution period, or not. The selectivity profile depends on the scheme used as well as on the number of protons attached to the heteronucleus. The selective HMBC experiment requires only a single echo sequence as no strict control of the signal phase is required. A complex glycoconjugate is used as a test compound for the new pulse sequences.     

Molecular hydrodynamics of inhomogeneous systems: The origin of slip boundary conditions

Molecular hydrodynamic equations derived by linear response theory for inhomogeneous systems are investigated in two extreme cases: a simple fluid in a gravitational field (an example of a long-range inhomogeneity), and a simple fluid in the presence of a rigid flat wall described by an infinite potential step (an example of a short-range inhomogeneity). In both cases the phenomenological equations result when the molecular equations are smoothed over a length which is large compared to the correlation lenght. For these systems the local equilibrium assumption is shown to be valid. In addition it is shown how the usual slip boundary conditions arise as a consequence of the interaction which causes the short-range inhomogeneity.     

Effects of long-range and homonuclear couplings on bilinear rotation pulses

Suppression of long-range heteronuclear interactions in J-resolved 2D NMR facilitates separate measurements of one-bond coupling constants. Analysis of the pulse sequence describes the efficiency of bilinear rotation in the presence of long-range couplings. It also predicts intensities of possible spurious responses. Theoretical and experimental evidence confirm that long-range couplings do not represent a serious obstacle against routine applications of spin manipulations in heteronuclear 2D NMR.     

局域共振型声子晶体中的缺陷态研究

本文以二维固体局域共振声子晶体为例,对次波长区域缺陷态的实现机理及其特点进行了探讨.众所周知,工作于次波长区域的声子晶体可以用有效媒质理论来描述,其色散关系对其构成单元的排列结构并不敏感,因而使得共振型声子晶体的带结构难以被其构成单元的局域空间无序所打破.本文发现共振型声子晶体在其带隙附近的色散关系可由共振单元间的长程相互作用来理解.基于这一理解,对目前文献中提出的两种实现局域态的方法进行了研究,结果表明缺陷态的引入正是通过打破这种长程相互作用来实现的.另外,在此理解的基础上,通过引入非各向同性"缺陷"共振单元,实现了纵波与横波导波模式的分离.     

2D TRIPLE in orientationally disordered samples--a means to resolve and determine relative orientation of hyperfine tensors

The two-dimensional (2D) TRIPLE experiment provides correlations between electron-nuclear double resonance (ENDOR) frequencies that belong to the same electron-spin manifold, M(S), and therefore allows to assign ENDOR lines to their specific paramagnetic centers and M(S) manifolds. This, in turn, also provides the relative signs of the hyperfine couplings. So far this experiment has been applied only to single crystals, where the cross-peaks in the 2D spectrum are well resolved with regular shapes. Here we introduce the application of the 2D TRIPLE experiment to orientationally disordered systems, where it can resolve overlapping powder patterns. Moreover, analysis of the shape of the cross-peaks shows that it is highly dependent on the relative orientation of the hyperfine tensors of the two nuclei contributing to this particular peak. This is done initially through a series of simulations and then demonstrated experimentally at a high field (W-band, 95 GHz). The first example concerned the (1)H hyperfine tensors of the stable radical alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) immobilized in a polystyrene matrix. Then, the experiment was applied to a more complex system, a frozen solution of Cu(II)-bis(2,2':6',2' terpyridine) complex. There, the 2D TRIPLE experiment was combined with the variable mixing time (VMT) ENDOR experiment, which determined the absolute sign of the hyperfine couplings involved, and orientation selective ENDOR experiments. Analysis of the three experiments gave the hyperfine tensors of a few coupled protons.     

Efficiency of purging sequences in the long-range heteronuclear shift correlation experiment

The efficiencies of a number of pulse sequences designed to remove directly bonded C-H correlations from long-range C-H shift correlation maps are evaluated. A two-step J filter sequence is shown to give good suppression in 1 D experiments. Its incorporation into the long-range C-H shift correlation experiment with a BIRD sequence at the center of the refocusing period gives the BIRDTRAP sequence, which is shown to yield 2D maps with a few very weak direct correlations and no artifacts. BIRDTRAP has a sensitivity higher than that of FLOCK.     

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.     

Use of changeover designs in subjective experiments

Experiments in which a number of subjects are asked to rate in some way a given set of stimuli are considered. In designing such experiments, the order in which the stimuli are presented to subjects is invariably taken into account both in the design and analysis. In many cases, the experimenter also presents the stimuli in such a way that each stimulus follows immediately after each of the other stimuli equally frequently. This is because a subject's response to a given stimulus may be influenced by the stimulus given in the immediately preceding period. However, the information available from arranging the stimuli in this manner is usually ignored in the statistical analysis of the experiment. The extraction of this information may be important and will often lead to a more precise experiment. In this paper these experiments are discussed and it is shown how they can be analysed so as to take into account this additional information. The technique is illustrated by an analysis of a subjective aircraft noise experiment.     

Measurement of one-bond heteronuclear dipolar coupling contributions for amine and diastereotopic methylene protons

One-bond heteronuclear and two-bond homonuclear residual dipolar couplings measured at methylene or amine sites can be utilized as long-range constraints in structure determination of molecules as well as to facilitate characterization of local conformation by stereospecific assignment of diastereotopic protons. We present two J-modulated HMQC type experiments to measure the one-bond heteronuclear dipolar coupling contributions of geminal protons individually. In addition two-bond homonuclear residual dipolar couplings between the diastereotopic protons are also obtained.