Application of biselective refocusing soft pulses to the simplification of heteronuclear correlation spectra

The biselective spin echo technique allows the signals of coupled proton pairs to be extracted from crowded liquid state proton NMR spectra. Its use as a preparation sequence in heteronuclear chemical shift correlation experiments requires the removal of the heteronuclear coupling interaction during the biselective echo time. The discrimination between coupled and uncoupled protons signals is achieved by double quantum filtration, which delivers antiphase magnetization states. The latter are not directly compatible with the design of an HSQC-like pulse sequence. The conversion of antiphase to in-phase magnetization states by a second biselective echo sequence solves this problem. The optimization of spin echo delays is also discussed. Lastly, the article presents modified HSQC and HMBC pulses sequences in which information is obtained solely for the biselectively selected proton pairs. A peracetylated trisaccharide was used as a test molecule.     

液体核磁共振 HSQC 实验的量子化学计算与模拟

核磁共振（NMR）异核单量子相干（HSQC）实验因具有较高的灵敏度和分辨率而被广泛用于液体大分子化合物的结构鉴定和研究．然而由于HSQC脉冲的复杂性,需要严格控制实验参数和实验条件才能得到高质量的谱图．本文基于量子力学原理对HSQC实验进行数学建模,通过理论推导、数值计算求解自旋1/2的IS双核体系在每个脉冲节点作用后的密度矩阵,然后结合二维NMR信号采样方法,使用计算机程序完成了该体系HSQC谱图的模拟,同时,还实现了乙醇分子的HSQC谱图模拟． HSQC实验的成功模拟基于对复杂演化过程的精确计算,可用于预测谱图以及实验参数改变对NMR谱图的影响,指导高质量HSQC实验谱图的采集．     

Fast imaging with the MMME sequence

The multiple-modulation-multiple-echo sequence, previously used for rapid measurement of diffusion, is extended to a method for single shot imaging. Removing the gradient switching requirement during the application of RF pulses by a constant frequency encoding gradient can shorten experiment time for ultrafast imaging. However, having the gradient on during the pulses gives rise to echo shape variations from off-resonance effects, which make the image reconstruction difficult. In this paper, we propose a simple method to deconvolve the echo shape variation from the true one-dimensional image. This method is extended to two-dimensional imaging by adding phase encoding gradients between echoes during the acquisition period to phase encode each echo separately. Slice selection is achieved by a frequency selective pulse at the beginning of the sequence. Imaging speed is mainly limited by the phase encoding gradients' switching times and echo overlap when echo spacing is very short. This technique can produce a single-shot image of sub-millimeter resolution in 5 ms.     

Analyses, extensions and comparison of three experimental schemes for measuring ((n)J(CH)+D(CH))-couplings at natural abundance

Three types of experiments for measuring (n)J(CH) heteronuclear long-range coupling constants are examined and extended with state-of-the-art pulse sequence building-blocks: The use of a HMBC with corresponding reference-HSQC for accurate coupling determination is combined with the constant time technique and the conversion of antiphase magnetization into ZQ/DQ-coherences; CPMG-based LR-CAHSQC and BIRD(r,X)-HSQMBC experiments are examined in detail with respect to their coherence transfer properties; finally, the HSQC-TOCSY-IPAP experiment is introduced, a sequence derived from previously published alpha and beta selective HSQC-TOCSYs using a different spin state selection technique and a recently developed ZQ-suppression method. The experiments are characterized with their advantages and disadvantages and compared using strychnine and menthol as standard molecules.     

Simplified multiplet pattern HSQC-TOCSY experiment for accurate determination of long-range heteronuclear coupling constants

A new two-dimensional pulse sequence for accurately determining heteronuclear coupling constants is presented. It is derived from HSQC and HECADE techniques with B0 gradient coherence selection. The main feature of the proposed method is spectra with only one component of the IS doublet; i.e., the final result is equivalent to a selective broadband excitation of either Salpha or Sbeta spin states and a preservation of these states during the entire experiment. The effect is obtained by an appropriate combination of in- and antiphase coherences. It is demonstrated that heteronuclear single-bond as well as long-range coupling constants and their relative signs are readily evaluated. The proposed sequence is equally or less sensitive to a variation of heteronuclear one-bond couplings than previously published, closely related sequences. The new method is applied to a peptide sample for determination of 3JN, Hbeta.     

Biselective refocusing pulses and the SERF experiment

The SERF experiment is a variant of the homonuclear J-resolved experiment, in which a single coupling constant is measured. It consists of a single chemical shift selective excitation that is followed by a biselective spin echo. Recent articles mention the existence of artefacts in SERF spectra that are supposedly related to pulse imperfections. This article presents a detailed study of the biselective refocusing pulses. It also reports a method for predicting the position and amplitude of the expected and unexpected 2D spectral peaks in SERF spectra. Artefacts can be partially eliminated by phase cycling or by the introduction of static field gradient pulses in the acquisition sequence. A procedure to obtain of pure absorption peaks in SERF spectra is proposed.     

Signal Enhancement Using 45° Water Flipback for 3D N-Edited ROESY and NOESY HMQC and HSQC

A novel implementation of the water flipback technique employing a 45° flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D ¹⁵N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45° water flipback are presented using both HMQC and HSQC for the ¹⁵N dimension. The 45° flipback pulse is followed by a gradient, a water selective 180° pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t₁ and t₂ evolution periods is suppressed using gradients. Water resonance planes from NOESY–HMQC and NOESY–HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein–water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.     

Signal enhancement using 45 degrees water flipback for 3D 15N-edited ROESY and NOESY HMQC and HSQC.

A novel implementation of the water flipback technique employing a 45 degrees flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D 15N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45 degrees water flipback are presented using both HMQC and HSQC for the 15N dimension. The 45 degrees flipback pulse is followed by a gradient, a water selective 180 degrees pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t1 and t2 evolution periods is suppressed using gradients. Water resonance planes from NOESY-HMQC and NOESY-HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein-water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.     

Measurement of longitudinal and rotating frame relaxation times through fully J-decoupled homonuclear spectra.

It is shown that fully J-decoupled homonuclear spectra involving Lorentzian lines can be readily obtained by straightforward processing of the 2D data arising from a conventional spin echo sequence (pi/2-t(1)/2-pi-t(1)/2-Acq(t(2))) used in the so-called J-resolved experiment. The method simply rests on power spectra with the drawback of lines having meaningless relative intensities. In principle, the experiment should also yield transverse relaxation times. Several tests demonstrate that this is not so, due to pulse imperfections and nonresolved long-range J couplings. Conversely, longitudinal and rotating frame relaxation times can be easily determined by means of an appropriate preparation period (for instance, a saturation-recovery period in the case of longitudinal relaxation) inserted before the 2D spin echo sequence. Since one is dealing with a single line per nucleus, relaxation measurements become reliable and accurate.     

Physical mechanisms of spin echoes. I. Two-pulse locked echo

The locked echo is the result of the application, to an inhomogeneously-broadened system, of a pulse sequence consisting of a short π/2 pulse, a free evolution period of length r and a long pulse with high turning angle (HTA). In this paper the nature and features of the signal detected after such a pulse sequence are reported upon by analysing the underlying physical mechanisms in the framework of the density operator formalism. It is found that the total signal does not contain any contribution from Free Induction Decay (FID)-like or anti-echo signals but comprises a group of several distinct echoes: the true simultaneous locked echo, arising from locked magnetization, and two or three non-simultaneous, oscillatory echoes similar to those observed in the single-pulse experiment. Properties of these echoes are deduced from the structure of the density operator at the end of the experiment. Extensive numerical simulations provide independent evidence of the correctness of the developed theory, display the variety of patterns shown by the locked echo when experimental conditions are changed, and also permit one to investigate the locked echo shape beyond the approximations introduced in the theory.     

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.     

Selective saturation in strongly inhomogeneous magnetic fields

The possibility to produce selective saturation by nuclear magnetic resonance (NMR) sequences of low-power radio-frequency pulses in strongly inhomogeneous magnetic fields is explored. The saturation of parts of the sensitive volume is produced by a particular pulse sequence with reduced amplitude distribution and the spectrum of the recorded signal is compared with the simulated spectrum. The spectra of the recorded free induction decays and echo signals are in good agreement with the simulated spectra of the pulse sequence, which demonstrates the effect of the selective saturation. The results obtained are an important step towards the development of new mobile and lowpower NMR equipments operating with inhomogeneous magnetic fields.     

Discrimination of C NMR signals in solid material with liquid-like behavior presenting residual dipolar proton–proton homonuclear interactions: application on seeds

In this communication, we propose, a modified spin echo fourier transform (SEFT) experiment run under magic angle spinning (MAS) to obtain structural informations of the liquid-like domains inside complex organic materials. It includes a proton-proton dipolar decoupling such as BLEW12 or Lee-Goldburg sequence just after the 180 degrees 13C refocusing pulse and short echo delays are used in order to overcome T(2) relaxation. This very easy implemented sequence allows a clear discrimination among fast relaxing 13C signals between those with a pure liquid-like behavior and those presenting residual proton-proton dipolar coupling. The interests of the sequence, combined with other classical NMR experiments, are illustrated on whole vegetable seeds that represent an example of a complex material.     

Discrimination of 13C NMR signals in solid material with liquid-like behavior presenting residual dipolar proton-proton homonuclear interactions: application on seeds

In this communication, we propose, a modified spin echo fourier transform (SEFT) experiment run under magic angle spinning (MAS) to obtain structural informations of the liquid-like domains inside complex organic materials. It includes a proton-proton dipolar decoupling such as BLEW12 or Lee-Goldburg sequence just after the 180 degrees 13C refocusing pulse and short echo delays are used in order to overcome T(2) relaxation. This very easy implemented sequence allows a clear discrimination among fast relaxing 13C signals between those with a pure liquid-like behavior and those presenting residual proton-proton dipolar coupling. The interests of the sequence, combined with other classical NMR experiments, are illustrated on whole vegetable seeds that represent an example of a complex material.     

Excitation conditions for quantitative determination of quadrupolar spins with one pulse or spin-echo sequences in solid-state NMR

One of the major problems concerning quadrupolar spins in solid-state NMR is their quantification. If the optimal excitation conditions with one radio-frequency pulse are widespread known now, this is not the case with the spin-echo sequences. This paper reports some theoretical predictions and their limitations concerning quantification with the echo obtained with spin-echo resonances. To realize that, first, the relative line intensity of a transition (m+1,m) is defined in order to allow the comparison of results, from different authors. Then results concerning one pulse excitation on a spinI=3/2 are summarized. The condition of short pulse excitation is generalized to higher spins using the Pauli matrices applied to the two extreme cases: hard pulse or non selective excitation, and selective excitation. Finally the same procedure has been followed for the spin-echo sequence involving twox-pulses. It was shown that the optimum conditions are: both the pulse length must be sufficiently short, and the interpulse delay should be taken as short as the duration of the FID provided the phase of the second pulse alternates without changing the receiver phase. In these conditions, the relative echo amplitude depends linearly on the first pulse length and quadratically on the second. The limitations are: the homonuclear magnetic dipolar interaction must be much smaller than the heteronuclear case which must be itself much smaller than the amplitude of the pulse. Furthermore, quantification with the echo requires the determination of the spin-spin relaxation time as well.     

Improved Spin-Echo-Edited NMR Diffusion Measurements

The need for simple and robust schemes for the analysis of ligand-protein binding has resulted in the development of diffusion-based NMR techniques that can be used to assay binding in protein solutions containing a mixture of several ligands. As a means of gaining spectral selectivity in NMR diffusion measurements, a simple experiment, the gradient modified spin-echo (GOSE), has been developed to reject the resonances of coupled spins and detect only the singlets in the (1)H NMR spectrum. This is accomplished by first using a spin echo to null the resonances of the coupled spins. Following the spin echo, the singlet magnetization is flipped out of the transverse plane and a dephasing gradient is applied to reduce the spectral artifacts resulting from incomplete cancellation of the J-coupled resonances. The resulting modular sequence is combined here with the BPPSTE pulse sequence; however, it could be easily incorporated into any pulse sequence where additional spectral selectivity is desired. Results obtained with the GOSE-BPPSTE pulse sequence are compared with those obtained with the BPPSTE and CPMG-BPPSTE experiments for a mixture containing the ligands resorcinol and tryptophan in a solution of human serum albumin.     

Cooling overall spin temperature: protein NMR experiments optimized for longitudinal relaxation effects

In experiments performed on protonated proteins at high fields, 80% of the NMR spectrometer time is spent waiting for the ¹H atoms to recover their polarization after recording the free induction decay. Selective excitation of a fraction of the protons in a large molecule has previously been shown to lead to faster longitudinal relaxation for the selected protons [K. Pervushin, B. Vögeli, A. Eletsky, Longitudinal ¹H relaxation optimization in TROSY NMR spectroscopy, J. Am. Chem. Soc. 124 (2002) 12898–12902; P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015; H.S. Attreya, T. Szyperski, G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment, Proc. Natl. Acad. Sci. USA 101 (2004) 9642–9647]. The pool of non-selected protons acts as a “thermal bath” and spin-diffusion processes (“flip-flop” transitions) channel the excess energy from the excited pool to the non-selected protons in regions of the molecule where other relaxation processes can dissipate the excess energy. We present here a sensitivity enhanced HSQC sequence (COST-HSQC), based on one selective E-BURP pulse, which can be used on protonated ¹⁵N enriched proteins (with or without ¹³C isotopic enrichment). This experiment is compared to a gradient sensitivity enhanced HSQC with a water flip-back pulse (the water flip-back pulse quenches the spin diffusion between ¹H^N and ¹H^α spins). This experiment is shown to have significant advantages in some circumstances. Some observed limitations, namely sample overheating with short recovery delays and complex longitudinal relaxation behaviour are discussed and analysed.     

High-resolution heteronuclear correlation spectroscopy based on spatial encoding and coherence transfer in inhomogeneous fields

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy has been proven to be a powerful technique for chemical, biological, and medical studies. Heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) are two frequently used 2D NMR methods. In combination with spatially encoded techniques, a heteronuclear 2D NMR spectrum can be acquired in several seconds and may be applied to monitoring chemical reactions. However, it is difficult to obtain high-resolution NMR spectra in inhomogeneous fields. Inspired by the idea of tracing the difference of precession frequencies between two different spins to yield high-resolution spectra, we propose a method with correlation acquisition option and J-resolved-like acquisition option to ultrafast obtain high-resolution HSQC/HMBC spectra and heteronuclear J-resolved-like spectra in inhomogeneous fields.