Single-scan NMR spectroscopy at arbitrary dimensions

Multidimensional nuclear magnetic resonance (NMR) provides one of the foremost analytical tools available to elucidate the structure and dynamics of complex molecules in their native states. Executing this kind of experiment generally requires collecting an n-dimensional time-domain signal S, from which the spectrum arises via an appropriate Fourier analysis of its various time variables. This time-domain signal is actually measured directly only along one of the time axes, while the effects introduced by the remaining time variables are monitored via a parametric incrementation of their values throughout independent experiments. Two-dimensional (2D) NMR experiments thus usually require longer acquisition times than unidimensional experiments, 3D NMR is orders-of-magnitude more time consuming than 2D spectroscopy, etc. Very recently, we proposed and demonstrated an approach whereby data acquisition in 2D NMR can be parallelized, enabling the collection of complete 2D spectral sets within a single transient. The present paper discusses the extension of this 2D NMR methodology to an arbitrary number of dimensions. The principles of the ensuing ultrafast n-dimensional NMR approach are described, and a variety of homo- and heteronuclear 3D and 4D NMR spectra collected within a fraction of a second are presented.     

Local covariance order diffusion-ordered spectroscopy: a powerful tool for mixture analysis

Diffusion-ordered spectroscopy (DOSY) is an important tool in NMR mixture analysis that has found use in most areas of chemistry, including organic synthesis, drug discovery, and supramolecular chemistry. Typically the aim is to disentangle the overlaid, and often overlapped, NMR spectra of individual mixture components and/or to obtain size and interaction information from their respective diffusion coefficients. The most common processing method, high-resolution DOSY, breaks down where component spectra overlap; here multivariate methods can be very effective, but only for small numbers (2-5) of components. In this study, we present a hybrid method, local covariance order DOSY (LOCODOSY), that breaks a spectral data set into suitable windows and analyzes each individually before combining the results. This approach uses a multivariate algorithm (e.g., SCORE or DECRA) to resolve only a small number of components in any given window. Because a small spectral region should contain signals from only a few components, even when the spectrum as a whole contains many more, the total number of resolvable chemical components rises dramatically. It is demonstrated here that complete resolution of component spectra can be achieved for mixtures that are much more complex than could previously be analyzed with DOSY. Thus, LOCODOSY is a powerful, flexible tool for processing NMR diffusion data of complex mixtures.     

Fast multidimensional localized parallel NMR spectroscopy for the analysis of samples

A parallel localized spectroscopy (PALSY) method is presented to speed up the acquisition of multidimensional NMR (nD) spectra. The sample is virtually divided into a discrete number of nonoverlapping slices that relax independently during consecutive scans of the experiment, affording a substantial reduction in the interscan relaxation delay and the total experiment time. PALSY was tested for the acquisition of three experiments 2D COSY, 2D DQF‐COSY and 2D TQF‐COSY in parallel, affording a time‐saving factor of 3–4. Some unique advantages are that the achievable resolution in any dimension is not compromised in any way: it uses conventional NMR data processing, it is not prone to generate spectral artifacts, and once calibrated, it can be used routinely with these and other combinations of NMR spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

Understanding J‐Modulation during Spatial Encoding for Sensitivity‐Optimized Ultrafast NMR Spectroscopy

Ultrafast (UF) NMR spectroscopy is an approach that yields 2D spectra in a single scan. This methodology has become a powerful analytical tool that is used in a large array of applications. However, UF NMR spectroscopy still suffers from an intrinsic low sensitivity, and from the need to compromise between sensitivity, spectral width, and resolution. In particular, the modulation of signal intensities by the spin–spin J‐coupling interaction (J‐modulation) impacts significantly on the intensities of the spectral peaks. This effect can lead to large sensitivity losses and even to missing spectral peaks, depending on the nature of the spin system. Herein, a general simulation package (Spinach) is used to describe J‐modulation effects in UF experiments. The results from simulations match with experimental data and the results of product operator calculations. Several methods are proposed to optimize the sensitivity in UF COSY spectra. The potential and drawbacks of the different strategies are also discussed. These approaches provide a way to adjust the sensitivity of UF experiments for a large range of applications.     

Sensitivity and lineshape improvement in ultrafast 2D NMR by optimized apodization in the spatially encoded dimension

Ultrafast 2D NMR allows the acquisition of a 2D spectrum in a single scan. However, even when the acquisition of ultrafast spectra is carried out under optimized conditions, the appearance and the sensitivity of 2D spectra are often not satisfactory compared with what one could expect from this promising methodology. This is due to limitations in terms of sensitivity, spectral width and resolution, and also to non-ideal lineshapes characterized by asymmetric sinc wiggles. Here, we identify the origin of these distortions by means of numerical simulations compared with experimental data. We then propose a processing approach to improve lineshapes while increasing the sensitivity of ultrafast experiments. The method consists in multiplying the Fourier transform of ultrafast echoes by an optimized apodization function. The principles of the method are described, and a variety of window functions are tested to determine optimum processing conditions. The approach is finally applied to ultrafast 2D spectra, leading to symmetric lineshapes with a sensitivity increased by a factor of 2.     

Microstructure determination of N-vinyl-2-pyrrolidone/butyl acrylate copolymers by NMR spectroscopy

The copolymer composition of N-vinyl-2-pyrrolidone/butyl acrylate (V/B) copolymers was determined from the quantitative ¹³C{¹H} NMR spectra. The monomer reactivity ratios for N-vinyl-2-pyrrolidone (V) and butyl acrylate (B) were found to be r_V=0.11±0.07, r_B=0.54±0.19, using the Kelen–Tudos and non-linear least-square error-in-variable (EVM) methods. The ¹³C{¹H} and ¹H NMR spectra of these copolymers are overlapping and complex. The complete spectral assignment of the carbon and proton NMR spectra were done by employing distortionless enhancement by polarization transfer (DEPT) and two-dimensional (2D) ¹³C–¹H heteronuclear single quantum correlation spectroscopy experiments. The 2D total correlation spectroscopy (TOCSY) (¹H–¹H homonuclear TOCSY) NMR spectrum was used to ascertain the various geminal and vicinal couplings in the copolymer.     

Progress in Hyperpolarized Ultrafast 2D NMR Spectroscopy

An important development in the field of NMR spectroscopy has been the advent of hyperpolarization approaches, capable of yielding nuclear spin states whose value exceeds by orders‐of‐magnitude what even the highest‐field spectrometers can afford under Boltzmann equilibrium. Included among these methods is an ex situ dynamic nuclear polarization (DNP) approach, which yields liquid‐phase samples possessing spin polarizations of up to 50 %. Although capable of providing an NMR sensitivity equivalent to the averaging of about 1 000 000 scans, this methodology is constrained to extract its “superspectrum” within a single—or at most a few—transients. This makes it a poor starting point for conventional 2D NMR acquisition experiments, which require a large number of scans that are identical to one another except for the increment of a certain t₁ delay. It has been recently suggested that by merging this ex situ DNP approach with spatially encoded “ultrafast” methods, a suitable starting point could arise for the acquisition of 2D spectra on hyperpolarized liquids. Herein, we describe the experimental principles, potential features, and current limitations of such integration between the two methodologies. For a variety of small molecules, these new hyperpolarized ultrafast experiments can, for equivalent overall durations, provide heteronuclear correlation spectra at significantly lower concentrations than those currently achievable by conventional 2D NMR acquisitions. A variety of challenges still remain to be solved before bringing the full potential of this new integrated 2D NMR approach to fruition; these outstanding issues are discussed.     

Probing Invisible,Excited Protein States by Non‐Uniformly Sampled Pseudo‐4D CEST Spectroscopy

Chemical exchange saturation transfer (CEST) NMR spectroscopy is a powerful tool for studies of slow timescale protein dynamics. Typical experiments are based on recording a large number of 2D data sets and quantifying peak intensities in each of the resulting planes. A weakness of the method is that peaks must be resolved in 2D spectra, limiting applications to relatively small proteins. Resolution is significantly improved in 3D spectra but recording uniformly sampled data is time‐prohibitive. Here we describe non‐uniformly sampled HNCO‐based pseudo‐4D CEST that provides excellent resolution in reasonable measurement times. Data analysis is done through fitting in the time domain, without the need of reconstructing the frequency dimensions, exploiting previously measured accurate peak positions in reference spectra. The methodology is demonstrated on several protein systems, including a nascent form of superoxide dismutase that is implicated in neurodegenerative disease.     

Targeted acquisition for real-time NMR spectroscopy

A target-oriented approach for the acquisition of information in biomolecular NMR spectroscopy is being developed. This approach combines concurrent data accumulation, processing, and monitoring of spectral quality. Real-time estimation of parameters allows acquisition to be stopped when results are complete and have a specified precision. The technique is based on multidimensional decomposition, which can process incomplete data. An incremental nonuniform sampling scheme ensures the optimization of resolution sensitivity. To validate this method, 3D HNCO spectra of three biomolecular systems (8 kDa ubiquitin, 22 kDa barstar-barnase complex, and 82 kDa malate synthase G) are processed incrementally at small acquisition time steps. The range of molecular sizes illustrates applicability in both sample- and sensitivity-limited regimes. In each case, the target was to acquire all backbone resonances in the spectra. For the three systems, the targets are achieved after 4.5 min, 1.6 h, and 22 h of acquisition time, respectively. A number of other targets that can be similarly monitored as a function of time are discussed.     

Hyperdimensional NMR spectroscopy with nonlinear sampling

An approach is described for joint interleaved recording, real-time processing, and analysis of NMR data sets. The method employs multidimensional decomposition to find common information in a set of conventional triple-resonance spectra recorded in the nonlinear sampling mode, and builds a model of hyperdimensional (HD) spectrum. While preserving sensitivity per unit of measurement time and allowing for maximal spectral resolution, the approach reduces data collection time on average by 2 orders of magnitude compared to the conventional method. The 7-10 dimensional HD spectrum, which is represented as a set of deconvoluted 1D vectors, is easy to handle and amenable for automated analysis. The method is exemplified by automated assignment for two protein systems of low and high spectral complexity: ubiquitin (globular, 8 kDa) and zetacyt (naturally disordered, 13 kDa). The collection and backbone assignment of the data sets are achieved in real time after approximately 1 and 10 h, respectively. The approach removes the most critical time bottlenecks in data acquisition and analysis. Thus, it can significantly increase the value of NMR spectroscopy in structural biology, for example, in high-throughput structural genomics applications.     

Analysis of laser-induced breakdown spectroscopy spectra: The case for extreme value statistics

In most instances, laser-induced breakdown spectroscopy (LIBS) spectra are obtained through analog accumulation of multiple shots in the spectrometer CCD. The average acquired in the CCD at a given wavelength is assumed to be a good representation of the population mean, which in turn is implicitly regarded to be the best estimator for the central value of the distribution of the spectrum at the same wavelength. Multiple analog accumulated spectra are taken and then in turn averaged wavelength-by-wavelength to represent the final spectrum. In this paper, the statistics of single-shot and analog accumulated LIBS spectra of both solids and liquids were examined to evaluate whether the spectrum averaging approach is statistically defensible. At a given wavelength, LIBS spectra are typically drawn from a Frechet extreme value distribution, and hence the mean of an ensemble of LIBS spectra is not necessarily an optimal summary statistic. Under circumstances that are broadly general, the sample mean for LIBS data is statistically inconsistent and the central limit theorem does not apply. This result appears to be due to very high shot-to-shot plasma variability in which a very small number of spectra are high in intensity while the majority are very weak, yielding the extreme value form of the distribution. The extreme value behavior persists when individual shots are analog accumulated. An optimal estimator in a well-defined sense for the spectral average at a given wavelength follows from the maximum likelihood method for the extreme value distribution. Example spectra taken with both an Echelle and a Czerny–Turner spectrometer are processed with this scheme to create smooth, high signal-to-noise summary spectra. Plasma imaging was used in an attempt to visually understand the observed variability and to validate the use of extreme value statistics. The data processing approach presented in this paper is statistically reliable and should be used for accurate comparisons of LIBS spectra instead of arithmetic averaging on either complete or censored data sets.     

Characterization of titanium dioxide nanoparticles dispersed in organic ligand solutions by using a diffusion-ordered spectroscopy-based strategy

Diffusion-ordered NMR spectroscopy (DOSY NMR) is presented as a tool for the determination of the diffusion coefficients of organic ligands in suspensions of titanium dioxide nanoparticles. The nanoparticles were prepared by a sol-gel process by hydrolysis and condensation reactions of titanium tetra-n-butoxide in the presence of pentane-2,4-dione (acacH: acetylacetone), as well as para-toluenesulfonic acid (pTsA) and n-butanol (nBuOH). NMR spectroscopic studies were performed in various deuterated solvents, on both dispersed xerosols and diluted sols. The bipolar-pulsed field gradient longitudinal eddy-current delay (LED) pulse sequence was used for data acquisition. The data were processed by inverse Laplace transformation (ILT), by using a maximum entropy algorithm, to afford 2D DOSY spectra. Different diffusion regimes for organic ligands in the bound and unbound states were successfully discriminated, more particularly in [D3]acetonitrile, thus allowing assessment of their interactions with the nanoparticles.     

Boosting the Resolution of 1H NMR Spectra by Homonuclear Broadband Decoupling

Broadband homonuclear decoupling of proton spectra, that is, the collapse of all multiplets into singlets, has the potential of boosting the resolution of ¹H NMR spectra. Several methods have been described in the last 40 years to achieve this goal. Most of them can only be applied in the indirect dimension of multi‐dimensional NMR spectra or special data processing is necessary to yield decoupled 1D proton spectra. Recently, complete decoupling of proton spectra during acquisition has been introduced; this not only significantly reduced the experimental time to record these spectra, but also removed the need for any sophisticated processing schemes. Here we present an introduction and overview of the techniques and applications of broadband proton‐decoupled proton experiments.     

Sparse sampling methods in multidimensional NMR

Although the discrete Fourier transform played an enabling role in the development of modern NMR spectroscopy, it suffers from a well-known difficulty providing high-resolution spectra from short data records. In multidimensional NMR experiments, so-called indirect time dimensions are sampled parametrically, with each instance of evolution times along the indirect dimensions sampled via separate one-dimensional experiments. The time required to conduct multidimensional experiments is directly proportional to the number of indirect evolution times sampled. Despite remarkable advances in resolution with increasing magnetic field strength, multiple dimensions remain essential for resolving individual resonances in NMR spectra of biological macromolecues. Conventional Fourier-based methods of spectrum analysis limit the resolution that can be practically achieved in the indirect dimensions. Nonuniform or sparse data collection strategies, together with suitable non-Fourier methods of spectrum analysis, enable high-resolution multidimensional spectra to be obtained. Although some of these approaches were first employed in NMR more than two decades ago, it is only relatively recently that they have been widely adopted. Here we describe the current practice of sparse sampling methods and prospects for further development of the approach to improve resolution and sensitivity and shorten experiment time in multidimensional NMR. While sparse sampling is particularly promising for multidimensional NMR, the basic principles could apply to other forms of multidimensional spectroscopy.     

High-resolution iterative frequency identification for NMR as a general strategy for multidimensional data collection

We describe a novel approach to the rapid collection and processing of multidimensional NMR data: "high-resolution iterative frequency identification for NMR" (HIFI-NMR). As with other reduced dimensionality approaches, HIFI-NMR collects n-dimensional data as a set of two-dimensional (2D) planes. The HIFI-NMR algorithm incorporates several innovative features. (1) Following the initial collection of two orthogonal 2D planes, tilted planes are selected adaptively, one-by-one. (2) Spectral space is analyzed in a rigorous statistical manner. (3) An online algorithm maintains a model that provides a probabilistic representation of the three-dimensional (3D) peak positions, derives the optimal angle for the next plane to be collected, and stops data collection when the addition of another plane would not improve the data model. (4) A robust statistical algorithm extracts information from the plane projections and is used to drive data collection. (5) Peak lists with associated probabilities are generated directly, without total reconstruction of the 3D spectrum; these are ready for use in subsequent assignment or structure determination steps. As a proof of principle, we have tested the approach with 3D triple-resonance experiments of the kind used to assign protein backbone and side-chain resonances. Peaks extracted automatically by HIFI-NMR, for both small and larger proteins, included approximately 98% of real peaks obtained from control experiments in which data were collected by conventional 3D methods. HIFI-NMR required about one-tenth the time for data collection and avoided subsequent data processing and peak-picking. The approach can be implemented on commercial NMR spectrometers and is extensible to higher-dimensional NMR.     

Monitoring Conformational Changes in an Enzyme Conversion Inhibitor Using Pure Shift Exchange NMR Spectroscopy

We report the acquisition of 2D NMR EXSY spectra with ultrahigh resolution, which allows for probing the slow conformational exchange process in a pharmaceutical compound. The resolution enhancement is achieved by implementing interferogram based PSYCHE homonuclear decoupling to generate a pure shift proton spectrum along the direct domain of the resulting data. The performance of this pure shift EXSY pulse sequence is compared to the standard experiment recorded under identical conditions. It is found that although being less sensitive and requiring a longer acquisition time, the quality of pure shift spectra allows for extracting exchange rates values that are coherent with the ones determined by standard approach, on a temperature range that demonstrates the robustness of the chosen homonuclear decoupling method. The resolution enhancement provided by the simplification of proton line shape allows for probing a higher number of proton sites whose analysis would have been biased using a standard method. These results open the way to a thorough and accurate study of chemical exchange processes based on a multi-site analysis of 2D pure shift EXSY spectra     

Conformation and protonation of 2,2'-bipyridine and 4,4'-bipyridine in acidic aqueous media and acidic ZSM-5 zeolites: a Raman scattering study.

In situ FT-Raman scattering spectroscopy was used to monitor the sorption kinetics of 2,2'- and 4,4'-bipyridine in acidic ZSM-5 zeolites. The data processing of all the Raman spectra was applied to extract the characteristic Raman spectra of occluded species and respective Raman contribution generated from many spectral data which resolves spectrum of mixture into pure component spectra without any prior information. The assignment of the extracted spectra was performed according to careful comparison with corresponding spectra extracted from a set of Raman spectra recorded during the protonation of 2,2'- or 4,4'-bipyridine (bpy) in hydrochloric acid aqueous solutions. The data processing of the Raman spectra recorded during the slow sorption of 4,4'-bpy in acidic H(n)ZSM-5 (n = 3, 6) zeolites provides specific Raman spectrum of N,N'-diprotonated dication 4,4'-bpyH(2)(2+) as unique species generated in the void space of acidic ZSM-5 zeolites. No evidence of Lewis acid sites was found during the sorption of 4,4'-bpy by Raman scattering spectroscopy. The data processing of the Raman spectra recorded during the slow sorption of 2,2'-bpy in acidic H(n)ZSM-5 (n = 3, 6) zeolites provides specific Raman spectrum of trans-N-monoprotonated cation 2,2'-bpyH+ as major species generated in the void space of acidic ZSM-5 zeolites at loading corresponding to 1 mol per unit cell. The trans/cis interconversion occurs at higher loading even after the complete uptake of the sorbate and indicates some rearrangement in the void space over a long time. The cations were found to be located in straight channels in the vicinity of the intersection with the zigzag channel of the porous materials with the expected conformations deduced from ab initio calculations. However, the motions of occluded species within the channel of ZSM-5 are hindered but remain in the range of the isotropic limit of a liquid at room temperature.     

Chemical‐Shift‐Resolved 19F NMR Spectroscopy between 13.5 and 135 MHz: Overhauser–DNP‐Enhanced Diagonal Suppressed Correlation Spectroscopy

Overhauser–DNP‐enhanced homonuclear 2D ¹⁹F correlation spectroscopy with diagonal suppression is presented for small molecules in the solution state at moderate fields. Multi‐frequency, multi‐radical studies demonstrate that these relatively low‐field experiments may be operated with sensitivity rivalling that of standard 200–1000 MHz NMR spectroscopy. Structural information is accessible without a sensitivity penalty, and diagonal suppressed 2D NMR correlations emerge despite the general lack of multiplet resolution in the 1D ODNP spectra. This powerful general approach avoids the rather stiff excitation, detection, and other special requirements of high‐field ¹⁹F NMR spectroscopy.     

Two-Dimensional Solid-State NMR Spectroscopy: New Possibilities for the Investigation of the Structure and Dynamics of Solid Polymers [New Analytical Methods (38)]

NMR spectroscopy is an effective method not only for examining liquid samples but also for characterizing molecular sturcture, order and dynamics in amorphous and ordered solids. Recent developments in the area of solid-state NMR spectroscopy span from model-dependent studies of conventional one-dimensional spectra to the more definitive two-dimensional (2D) spectra which provide more specific information. For example, with 2D-NMR spectroscopy it is possible to determine the orientational distribution functions of molecular segments in drawn polymers and to distinguish different mechanisms of complex molecular motions. Following an introduction to basic NMR spectroscopy, an overview of the current state-of-the-art of 2D methods in solid-state NMR spectroscopy is presented and demonstrated with selected examples.     

NMR Backbone Assignment of Large Proteins by Using 13Cα‐Only Triple‐Resonance Experiments

Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical‐shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency degeneracy of the ¹³C_α nuclei. Herein, we present a covariance NMR strategy to assign the backbone chemical shifts by using only HN(CO)CA and HNCA spectra that has a high sensitivity even for large proteins. By using the peak linear correlation coefficient (LCC), which is a sensitive probe even for tiny chemical‐shift displacements, we correctly identify the fidelity of approximately 92 % cross‐peaks in the covariance spectrum, which is thus a significant improvement on the approach developed by Snyder and Brüschweiler (66 %) and the use of spectral derivatives (50 %). Thus, we calculate the 4D covariance spectrum from HN(CO)CA and HNCA experiments, in which cross‐peaks with LCCs above a universal threshold are considered as true correlations. This 4D covariance spectrum enables the sequential assignment of a 42 kDa maltose binding protein (MBP), in which about 95 % residues are successfully assigned with a high accuracy of 98 %. Our LCC approach, therefore, paves the way for a residue‐by‐residue study of the backbone structure and dynamics of large proteins.