Extending the limits of the selective 1D NOESY experiment with an improved selective TOCSY edited preparation function

Compared to its 2D counterpart, the selective 1D NOESY experiment offers greatly simplified spectral interpretation and is invaluable to the structure elucidation of small-to-medium sized molecules, although its application is limited to well-resolved resonances only. The doubly selective 1D TOCSY-NOESY experiment allows the 1D NOESY experiment to be extended to resonances within overlapped spectral regions. However, existing methods do not address the critical issue of zero-quantum interference, which leads to severe anti-phase distortions to the line shape of scalar coupled spins and often complicates the identification of weak NOE enhancements. In this communication, we describe an improved selective TOCSY edited preparation (STEP) function and its application to the selective 1D NOESY experiment. The STEP function incorporates a novel zero-quantum filter introduced by Thrippleton and Keeler [Angew. Chem. Int. Ed. 42 (2003) 3938], which permits essentially complete suppression of zero-quantum coherence in a single scan. Residual anti-phase distortions due to spin-state mixing are removed using the double difference methodology reported by Shaka et al. [45th Experimental NMR Conference, Pacific Grove, USA, 2004]. The combined use of these techniques ensures that the final spectra are free of distortions, which is crucial to the reliable detection of weak NOE enhancements. Although employed as an additional preparation period in the example demonstrated here, the STEP function affords a general editing tool for spectral simplification and can be applied to a range of experiments.     

High resolution 13C DOSY: the DEPTSE experiment

High Resolution Diffusion-ordered Spectroscopy (HR-DOSY) is a valuable tool for mixture analysis by NMR. It separates the signals from different components according to their diffusion behavior, and can provide exquisite diffusion resolution when there is no signal overlap. In HR-DOSY experiments on (1)H (by far the most common nucleus used for DOSY) there is frequent signal overlap that confuses interpretation. In contrast, a (13)C spectrum usually has little overlap, and is in this respect a much better option for a DOSY experiment. The low signal-to-noise ratio is a critical limiting factor, but with recent technical advances such as cryogenic probes this problem is now less acute. The most widely-used pulse sequences for (13)C DOSY perform diffusion encoding with (1)H, using a stimulated echo in which half of the signal is lost. This signal loss can be avoided by encoding diffusion with (13)C in a spin echo experiment such as the DEPTSE pulse sequence described here.     

Resolving overlaps in diffusion encoded spectra using band-selective pulses in a 3D BEST-DOSY experiment

A novel diffusion-edited 3D NMR experiment that incorporates a BEST-HMQC pulse sequence in its implementation is presented. Heteronuclear 3D DOSY NMR experiments are useful in elucidating the diffusion coefficients of individual constituents of a mixture, especially in cases where the proton NMR 2D DOSY spectra show considerable overlap. The present 3D BEST-DOSY pulse sequence provides a more sensitive and less time-consuming alternative to standard 3D HMQC-DOSY experiments. Cleanly separated subspectra of individual mixture components are obtained, leading to the determination of diffusion coefficients with better accuracy. The feasibility of the technique is demonstrated on a mixture of amino acids, on a mixture of small molecules with similar diffusion coefficients, and on a complex mixture with large dynamic range (commercial gasoline). The implications of using adiabatic decoupling schemes and band-selective shaped pulses for selective BEST-DOSY experiments on proteins are also discussed.     

弱耦合自旋1/2二核系统二维动态NMR谱

二维动态NMR谱通常可由三脉冲实验得到。但如果核系统有标量耦合,就有可能混淆实验所得的信息。本文在Liouvelle空间中求出二核弱耦合系统的二维理论动态谱,给出AX(I=1/2)系统中的密度矩阵的解析解。文中指出:(1)在梯度脉冲作用之后,密度矩阵将留下z分量、零量子相干和双量子相干。(2)标量耦合与交换作用、弛豫作用的影响不同。前者只对零量子相干及双量子相干有贡献。如果实验得当,有希望消除标量耦合对动态谱的干扰。 关键词：     

Removal of zero-quantum peaks from 1D selective TOCSY and NOESY spectra

Selective 1D TOCSY and NOESY experiments are widely used for structure determination. However, they often give distorted peak shapes owing to coherence transfer though zero-quantum coherence (ZQ) which cannot be suppressed by conventional phase cycling or pulse-field gradients. This paper demonstrates that ZQ contributions can be removed from selective 1D spectra by introducing a ZQ evolution time, as previously demonstrated for 2D NOESY spectra by Wang et al. [A three-dimensional method for the separation of zero-quantum coherence and NOE in NOESY spectra, J. Magn. Reson. A 102 (1993) 116-121]. This approach is simple to implement and robust, and is not demanding of spectrometer hardware. Using a new approach to phase cycling described here, spectra can be acquired in a similar time to spectra without a ZQ evolution time.     

High-resolution absorptive intermolecular multiple-quantum coherence NMR spectroscopy under inhomogeneous fields

Intermolecular multiple-quantum coherence (iMQC) is capable of improving NMR spectral resolution using a 2D shearing manipulation method. A pulse sequence termed CT-iDH, which combines intermolecular double-quantum filter (iDQF) with a modified constant-time (CT) scheme, is designed to achieve fast acquisition of high-resolution intermolecular zero-quantum coherences (iZQCs) and intermolecular double-quantum coherences (iDQCs) spectra without strong coupling artifacts. Furthermore, double-absorption lineshapes are first realized in 2D intermolecular multi-quantum coherences (iMQCs) spectra under inhomogeneous fields through a combination of iZQC and iDQC signals to double the resolution without loss of sensitivity. Theoretically the spectral linewidth can be further reduced by half compared to original iMQC high-resolution spectra. Several experiments were performed to test the feasibility of the new method and the improvements are evaluated quantitatively. The study suggests potential applications for in vivo spectroscopy.     

Gradient- and Sensitivity-Enhanced TOCSY Experiments

A pulsed field gradient version of the sensitivity-enhanced 2D TOCSY experiment is proposed which yields high-quality spectra with improved sensitivity and a minimum of two scans pert₁increment. For rapid acquisition of 1D TOCSY spectra, the 1D DPFGSE–TOCSY experiment was modified to include phase-encoded multiple-selective excitation followed by a simple spectral editing. Combination of these two building blocks is used in a sensitivity-enhanced 2D analog of the 3D TOCSY–TOCSY experiment which provides an efficient tool for resolving severely overlapped signals of oligomers in short experimental time.     

Improving pulse sequences for 3D DOSY: convection compensation

Signal overlap in the NMR dimension significantly complicates the construction and analysis of 2D diffusion-ordered (DOSY) spectra. Such problems can often be reduced or even eliminated by extending the NMR domain of a DOSY experiment into two dimensions, giving a 3D-DOSY spectrum. To date such experiments have generally sacrificed some signal-to-noise ratio and have required extensive and time-consuming phase cycling. A new family of pulse sequences with internal diffusion encoding (IDOSY) has been introduced which avoids both of these problems. It is often straightforward to incorporate convection compensation in such sequences at no cost in signal-to-noise ratio. Here, some of the problems caused by convection in DOSY are described and illustrated, and the efficacy of convection compensation in the 2DJ-IDOSY and COSY-IDOSY experiments is demonstrated.     

High resolution heteronuclear correlation NMR spectroscopy between quadrupolar nuclei and protons in the solid state

A high resolution two-dimensional solid state NMR experiment is presented that correlates half-integer quadrupolar spins with protons. In this experiment the quadrupolar nuclei evolve during t1 under a split-t1, FAM-enhanced MQMAS pulse scheme. After each t1 period ending at the MQMAS echo position, single quantum magnetization is transferred, via a cross polarization process in the mixing time, from the quadrupolar nuclei to the protons. High-resolution proton signals are then detected in the t2 time domain during wPMLG5* homonuclear decoupling. The experiment has been demonstrated on a powder sample of sodium citrate and 23Na-1H 2D correlation spectra have been obtained. From the HETCOR spectra and the regular MQMAS spectrum, the three crystallographically inequivalent Na+ sites in the asymmetric unit were assigned. This MQMAS-wPMLG HETCOR pulse sequence can be used for spectral editing of half-integer quadrupolar nuclei coupled to protons.     

扩散序谱(DOSY)实验中扩散系数维数字分辨率的影响

核磁共振二维扩散序谱（DOSY）实验测定溶液中分子自扩散系数时,扩散系数维的数据点及其数字分辨率直接影响了测定值的精度.在较系统地确定了DOSY实验本身偏差范围的基础上,本文研究了扩散系数维不同的数字分辨率对测定值的影响,包括其引入偏差的来源以及形成偏差的大小.由于不同的溶液条件下分子扩散系数的改变可直接用于表征分子结构或状态的变化,本文提出的相对数字分辨率与扩散系数相对变化值的直接比较,可直观地表明数字分辨率对扩散系数测定值的影响.     

Double-quantum excitation in the NMR of spinning solids by pulse-assisted rotational resonance

We describe a new technique for double-quantum excitation in magic-angle-spinning NMR of powdered solids. The technique is designed to efficiently excite double-quantum coherence in the vicinity of a rotational resonance condition. The offset from rotational resonance allows the double-quantum filtered signals to be observed with high resolution and sensitivity. The method uses rotational excitation of zero-quantum coherence, assisted by radiofrequency pulse cycles. The zero-quantum coherence is converted into double-quantum coherence by a frequency-selective inversion sequence. Experiments on [(13)C(2), (15)N]-glycine demonstrate a double-quantum filtering efficiency of approximately 41% at a sample rotation frequency of 8.300 kHz, which is 1.600 kHz away from the n = 1 rotational resonance. We achieve 32% double-quantum filtering efficiency at a spinning frequency of 9.250 kHz, which is 2.550 kHz away from rotational resonance.     

STD-DOSY: A new NMR method to analyze multi-component enzyme/substrate systems

A new approach to analyze multi-component Saturation Transfer Difference (STD) NMR spectra by combining the STD and the DOSY experiment is proposed. The resulting pulse sequence was successfully used to simplify an exemplary multi-component protein/substrate system by means of standard DOSY processing methods. Furthermore, the same experiment could be applied to calculate the ratio of saturated substrate molecules and its saturation rate in the case of competitive interactions. This ratio depends on the strength of this interaction between the substrates and the protein, so that this kind of information could be extracted from the results of our experiment.     

Two-dimensional DOSY experiment with Excitation Sculpting water suppression for the analysis of natural and biological media

The Bipolar Pulse Pair Stimulated Echo NMR pulse sequence was modified to blend the original Excitation Sculpting water signal suppression. The sequence is a powerful tool to generate rapidly, with a good spectrum quality, bidimensional DOSY experiments without solvent signal, thus allowing the analysis of complex mixtures such as plant extracts or biofluids. The sequence has also been successfully implemented for a protein at very-low concentration in interaction with a small ligand, namely the salivary IB5 protein binding the polyphenol epigallocatechine gallate. The artifacts created by this sequence can be observed, checked and removed thanks to NPK and NMRnotebook softwares to give a perfect bidimensional DOSY spectrum.     

J-modulation effects in DOSY experiments and their suppression: the Oneshot45 experiment

Diffusion-ordered spectroscopy (DOSY) is a powerful NMR method for identifying compounds in mixtures. DOSY experiments are very demanding of spectral quality; even small deviations from expected behaviour in NMR signals can cause significant distortions in the diffusion domain. This is a particular problem when signals overlap, so it is very important to be able to acquire clean data with as little overlap as possible. DOSY experiments all suffer to a greater or lesser extent from multiplet phase distortions caused by J-modulation, requiring a trade-off between such distortions and gradient pulse width. Multiplet distortions increase spectral overlap and may cause unexpected and misleading apparent diffusion coefficients in DOSY spectra. These effects are described here and a simple and effective remedy, the addition of a 45° purging pulse immediately before the onset of acquisition to remove the unwanted anti-phase terms, is demonstrated. As well as affording significantly cleaner results, the new method allows much longer diffusion-encoding pulses to be used without problems from J-modulation, and hence greatly increases the range of molecular sizes that can be studied for coupled spin systems. The sensitivity loss is negligible and the added phase cycling is modest. The new method is illustrated for a widely-used general purpose DOSY pulse sequence, Oneshot.     

A homodecoupled diffusion experiment for the analysis of complex mixtures by NMR

Diffusion ordered spectroscopy (DOSY) relies on differences in translation diffusion as a means to separate components in a solution mixture. However, the analysis of spectra of mixtures can be problematic because spectral overlap. It is the aim of this article to propose a pulse sequence and processing method that leads to a complete 2D homodecoupled-DOSY experiment. This experiment offers several advantages that could extend the range of applications to more complex mixtures by achieving important improvements in both signal dispersion and sensitivity.     

Amino acid type identification in NMR spectra of proteins via beta- and gamma-carbon edited experiments

In this work, we introduce a set of pulse sequences that provide amino acid type identification of the NH correlation signals of proteins. The first pulse sequence is a modification of the CBCA(CO)NH experiment that exploits spin-coupling topologies to differentiate between amino acid types. A set of eight 2D ¹H–¹⁵N correlation spectra is recorded where the sign of the cross-peaks change from one spectrum to another according to the amino acid type of the preceding residue in the protein sequence. Linear combination of these eight data sets produces four subspectra. Taking also into account the sign of the correlation signals, this method allows the classification of the NH signals into six different groups, depending on the character of the preceding residue. This sequence is complemented with a (CGCBCACO)NH experiment that allows the subdivision of the largest of these groups into two smaller ones. Finally, a modification of the CBCANH experiment led to a similar classification of NH signals into six different groups, but now depending on the type of its own amino acid. The set of pulse sequences is demonstrated with two proteins of small to moderate size.     

Rapid high-resolution four-dimensional NMR spectroscopy using the filter diagonalization method and its advantages for detailed structural elucidation of oligosaccharides

Four-dimensional nuclear magnetic resonance spectroscopy with high resolution of signals in the indirect dimensions is reported as an implementation of the filter diagonalization method (FDM). Using an oligosaccharide derivatized with 13C-labeled acetyl isotags, a four-dimensional constant-time pulse sequence was tailored for conjoint use with the FDM. Results demonstrate that high resolution in all dimensions can be achieved using a relatively short experimental time period (19 h), even though the spectrum is highly congested in the direct and all three indirect dimensions. The combined use of isotags, constant-time pulse sequences, and FDM permits rapid isolation of sugar ring proton spin systems in multiple dimensions and enables all endocyclic J-couplings to be simply measured, the key goal to assigning sugar stereochemistry and anomeric configuration. A general method for rapid, unambiguous elucidation of spin systems in oligosaccharides has been a long-sought goal of carbohydrate NMR, and isotags combined with the FDM now enable this to be easily performed. Additional general advantages of the FDM program for generating high-resolution 2D slices in any dimension from a 4D spectrum are emphasized.     

Frequency-domain Hadamard spectroscopy

A new technique is proposed for multichannel excitation and detection of NMR signals in the frequency domain, an alternative to the widely used pulse-excited Fourier transform method. An extensive array of N radiofrequency irradiation channels covers the spectrum of interest. A selective radiofrequency pulse sequence is applied to each channel, generating a steady-state NMR response acquired one-point-at-a-time in the intervals between pulses. The excitation pattern is repeated N times, phase-encoded according to a Hadamard matrix, and the corresponding N composite responses are decoded by reference to the same matrix. This multiplex technique offers the same sensitivity advantage as conventional Fourier transform spectroscopy. The irradiation pattern may be tailored to concentrate on interesting spectral regions, to facilitate homonuclear double resonance, or to avoid exciting strong solvent peaks. As no free induction decay is involved, the new method avoids problems of pulse breakthrough or lineshape distortion by premature termination of the time-domain signal.     

Heteronuclear intermolecular single-quantum coherences in liquid nuclear magnetic resonance

This paper analyses the heteronuclear Cosy Revamped by Asymmetric Z-gradient Echo Detection pulse sequence. General theoretical expressions of the pulse sequence with arbitrary flip angles were derived by using dipolar field treatment and signals originating from heteronuclear intermolecular single-quantum coherences （iSQCs） in highly-polarized two spin-1/2 systems were mainly discussed in order to find the optimal flip angles. The results show that signals from heteronuclear iSQCs decay slower than those from intermolecular double-quantum coherences or intermolecular zero-quantum coherences. Magical angle experiments validate that the signals are from heteronuclear iSQCs and insensitive to the imperfection of radio-frequency flip angles. All experimental observations are in excellent agreement with theoretical predictions. The quantum-mechanical treatment leads to similar predictions to the dipolar field treatment.     

Solvent-localized NMR spectroscopy using the distant dipolar field: a method for NMR separations with a single gradient

Solvent-localized NMR (SOLO) is a new method which allows the separation of NMR spectra of substances dissolved in different solvents. It uses the selective HOMOGENIZED pulse sequence to produce a two-dimensional NMR spectrum resulting from intermolecular zero-quantum coherences in one distinct solvent. The detected signal is locally refocused by the action of the distant dipolar field, which is created by a frequency selective pulse only in regions containing the selected solvent. The prerequisites are that the different solvents have sufficiently different chemical shifts to be excited separately and that compartments with different solvents are spatially separated by more than the typical diffusion distance. Here, the method is demonstrated for the solvents water and DMSO on a length scale of 0.5 mm. Because signal in the spectra is refocused locally, SOLO is insensitive to variations in the magnetic field which may result from inhomogeneities or structures in the sample. This makes applications in strongly structured samples possible. SOLO is the first method that achieves localization of NMR signal with a single gradient pulse. Therefore, it can be used in conventional NMR spectrometers with one-axis gradient systems and lends itself to a wide range of applications including in vivo NMR.